Protein Folding and Expression Folding, Expression and Analysis
Contents 1. Introduction.......................................................................................................... 3
Contents
2. Expression Systems................................................................................................ 4 Human Cell-Free Expression System.......................................................................................................................................4 Brevibacillus Expression System II............................................................................................................................................6 B. subtilis Secretory Protein Expression System II...............................................................................................................8 3. Products for Increased Protein Yield and Purity..................................................... 9 pCold Expression Vectors.............................................................................................................................................................9 pCold TF Vector............................................................................................................................................................................ 10 pCold™ ProS2 DNA...................................................................................................................................................................... 10 mRNA Interferase™-MazF Enzyme......................................................................................................................................... 10 SPP System..................................................................................................................................................................................... 11 4. Mammalian Expression Vectors.............................................................................12 pBApo-CMV Vectors.................................................................................................................................................................... 12 pBApo-EF1a................................................................................................................................................................................... 12 pDON-AI-2....................................................................................................................................................................................... 13 pMEI-5.............................................................................................................................................................................................. 13 pDON-5............................................................................................................................................................................................ 14 5. Folding.................................................................................................................15 Chaperone Plasmid Set.............................................................................................................................................................. 15 Chaperonin GroE.......................................................................................................................................................................... 16 Corystein™ (Purothionin) Reagent......................................................................................................................................... 16 Refolding CA Kit............................................................................................................................................................................ 16 6. Application Notes.................................................................................................17 High-level Secretion of Recombinant Protein using the Brevibacillus Expression System.............................. 17 The pCold TF Protein Expression System Produces Soluble, Active Protein in E. coli........................................ 20 Unfolding the Potential of Proteins...................................................................................................................................... 22 7. FAQs.....................................................................................................................24 SPP System™ (Single Protein Production System)........................................................................................................... 24 pCold Expression Vectors.......................................................................................................................................................... 25 Refolding CA Kit............................................................................................................................................................................ 26 Chaperone Plasmid Set.............................................................................................................................................................. 27 8. Protein Sequencing and Analysis Products............................................................28 9. High Fidelity PCR Enzymes....................................................................................29 10. Takara Related Products.......................................................................................30 11. Clontech Related Products....................................................................................31
2
Clontech, A Takara Bio Inc. Company • www.clontech.com/takara
Introduction
Takara’s Protein Products Protein Folding Products
Chaperone Assisted Folding
Chaperonin GroE
Increase Level of Active Protein
Corystein™ Reagent Reforming Disulfide Bonds
Chaperone Plasmid Set Maximize Soluble Active Protein
Protein Expression Products
Insoluble Protein or Protein Toxic to Cell
Refolding CA Kit Optimize Refolding of Inclusion Bodies
Increase Protein Yield and Purity
pCold Vectors
Increase Recombinant Protein Yield
pCold TF DNA
Increase Expression using Trigger Factor
pCold Pro S2
Express Soluble Fusion Proteins
SPP System™ Kits
Single Protein Production System
Mammalian Expression
pBApo-CMV Vectors
All-purpose Gene Expression Vectors
High Yield Active Protein
Human Cell Free Expression System High Efficiency Cell-free Protein Production
pBApo-EF1α Vectors
Brevibacillus Expression System II
pDON-AI-2 Vectors
B. subtilus Expression System
Strong promoter for increased expression
Retroviral Vector for High-efficiency Gene Transduction
High Efficiency Protein Production
Secretory Protein Production
pMEI-5 Vectors
Retroviral Vector for High Expression
mRNA Interferase™ Plasmid MazF Enzyme
pDON-5 Vector
Retroviral Vector for High-efficiency Gene Transduction and High Expression
Active Protein
Ready for purification and/or analysis
Clontech, A Takara Bio Inc. Company • www.clontech.com/takara
3
1 Introduction
Protein expression is not an easy process; however, it can be made easier with the help of products that aid expression and folding. There are many factors which can prohibit the production of functional protein. Often it is not possible to predict which factors may impact expression of a particular protein of interest. In order for production of your target protein to be successful, map the process out from the beginning. The expression process consists of four steps: cloning, expression, purification, and characterization. Each step can be optimized to obtain a sufficient amount of functional protein. Consider the end use of the protein. Is it for biochemical studies or for structural analysis (NMR, X-ray crystallography)? The answer should help determine the best production process for your needs. Takara Bio offers a wide selection of tools to aid every step of the protein production process.
pT7-IRES His-N DNA
Human Cell-Free Expression System
Expression Systems
2
Code No. 3290 Shipping at － 20℃ Human Cell-Free Protein Expression System 3281 Size: 20 μg Store at － 20℃ pT7-IRES His-N DNA 3290 pT7-IRES His-C DNA 3291 ＊ 2 years from date of receipt under proper storage pT7-IRES Myc-N DNA 3292 conditions.
10 rxns 20 µg 20 µg Usage : 20 µg Protein expression using Human Cell-Free Protein Expression System.
Features
Vector map for pT7-IRES His-N DNA :
Lot No.
Concentration : as 1 h in a single-tube reaction 0.5 μg/μl • Easy-to-use system allows generation of protein in as little 40 μl • Provides higher yield of functionalVolume protein and: greater consistency than rabbit reticulocyte or wheat germ in vitro translation systems • Excellent with challenging proteins such as large proteins (over 150 kDa) and proteins requiring post-translational modification Regarding protocol for protein expression, please • Amenable to high-throughput screening; bulkthe sizes available. refer to product manual for Human Cell-Free Pro-
Applications
tein Expression System (Cat. #3281).
Purity : 1． Confirmed to maintain the region from T7 promoter to T7 terminator by dideoxy sequencing method. 2． Shown to be cleaved at a single site by Eco R I and at two sites by Hin d III.
polyA
T7 terminator
Multiple Cloning Site Factor Xa Site His-Tag EMCV IRES
Ampr
pT7-IRES His-N DNA (3,429 bp)
: • Expression of toxic proteins that areDescription lethal to host cells of in vivo expression systems pT7-IRES DNA series are expression vectors designed for Human Cell• Rapid analysis of protein function Free Protein Expression System. Tag sequence such as His-Tag or c-Myc ColE1 ori Tag, Factor Xa cleavage site, Multiple Cloning Site (MCS), polyA signal, • Rapid analysis of mutation series orandtruncation series: quickly and T7 terminator are generate located at protein the downstream of T7 promoter and EMCV IRES. There are vectors with some arrangements for the kind of Tag assess functionality using your downstream assay Figure 1. Map of pT7-IRES vector. and the location. • High-throughput proteomic studiespT7-IRES His-N DNA is an expression vector including His-Tag sequence. Using pT7-IRES His-N DNA together with Human Cell-Free Protein Expression System (Cat. #3281) enables the expression • Expression of proteins that are easily degraded or insoluble in conventional in vivo of your target as N-terminal fusion of His-Tag. Factor Xa cleavage site is inserted expression systems such as E. coli protein so that His-Tag can be removed from the expressed fusion protein. Note pT7-IRES Vectors: Target gene cloned into MCS in frame is transcribed as RNA-containingThis product is for research use only. It is not intended for use in Each vector provides 20 or µgdiagnostic DNA at a procedures concentration of 0.5 µg/µL (40 µL total therapeutic for humans or animals. Also, do volume). EMCV IRES under the control of T7 promoter. By the effect of EMCV IRES, Description designed to promote protein translation initiation, efficient high level
not use this product as food, cosmetic, or household item, etc. Takara products may not be resold or transferred, modified for resale
expression be Takara performed The Human Cell-Free Protein Expression Systemcan from BioinisHuman easy toCell-Free use. TheProtein Expression transfer, or used to manufacture commercial products without pT7-IRESor Vector Information System. written approval from TAKARA BIO INC. single-tube reaction is easily assembled and protein synthesis is complete in as little as 1 If you require licenses for other use, please contact us by phone at Vectors in the pT7-IRES series T7 promoter and EMCV IRES sequence to Form : 10 mM Tris-HCl, pH8.0 +81 77 543 7247 orinclude from oura website at www.takara-bio.com. h at 32°C. The Human Cell-Free Protein Expression System provides high yield (e.g., Your use of this product is also subject to compliance with any 1 mM EDTA facilitate transcription and translation in the Human Protein Expression System, applicable licensing requirements describedCell-Free on the product web 50 µg/ml of human eIF4G) of functional protein, including proteins requiring page. It isCloning your responsibility toconvenient review, understand and adhere to His-tag or Myc as well as a Multiple Site (MCS), tags (Nor C-terminal Preparation : Purified by ion-exchange column. modifications such as glycosylation, phosphorylation, or fatty acylation. Excellent yield any restrictions imposed by such statements. tag sequences), a Factor Xa protease cleavage site for tag removal, poly-A signal, and T7 is observed even with large proteins (over kDa). Genes v201107Da Chain150 length : 3,249 bp of interest may be cloned terminator. rapidly into pT7-IRES vectors using In-Fusion cloning technology. After expression, MCS :
proteins with N-terminal or C-terminal His-tag or N-terminal Myc tag can Nco be generated, I depending on choice of vector. Bulk sizesEMCV are available for high-throughput studies; IRES Nhe I 5’- T AACGT・・・・・・T AA T A TGGCCACAACC ATG GC T AGC contact
[email protected] for more information.
• pT7-IRES His-N DNA (Cat. # 3290) encodes an N-terminal His-tag Nde I Sac I Xho I • pT7-IRES His-C DNAFactor (Cat.Xa# 3291) encodes a C-terminal His-tag CAC CA T CAC CA T CAC CA T AT C GAA GGG CGC CAT AT G GAG CT C CT C GAG 3’-A T TGCA・・・・・・A T T A T ACCGGTGT TGG T AC CGA T CG GTG GT• A GTG GT A GTG GT A T AG C T T# CCC GT A T an AC N-terminal C T C GAG GAG C T Ctag pT7-IRES Myc-N DNA (Cat. 3292)GCG encodes c-Myc MetforAla In vitro translation has many advantages for protein expression: it is excellent rapidSer His His His His His His Ile Glu Gly Arg His Met Glu Leu Leu Glu c II Storage studies of protein function or features, amenable to high-throughputHinstudies, useful for Bam H I Eco R I Spe I Pst I Xba I End Sal I proteins that are degraded or insoluble with in vivo systems, and can be used to generate Cell-Free Protein Expression System (Cat. # 3281): –80°C GT AA T C-3’ GGA T CC GAA T T C AC T AGT GT C GAC C TG CAG T C T AGA T• AGHuman lethal proteins that cannot be expressed systems toxicity. In contrast CC Tusing AGG in C Tvivo T AAG TGA Tdue CA to CAG C TG GAC GT C AGA T C T A T C CA T T AG-5’ • pT7-IRES Vectors (Cat. #s 3290, 3291, 3292): –20°C Gly inSer Phe Thr systems Ser Valalso Asp Leu postGln Ser Arg to expression using prokaryotic host cells, vitroGlutranslation allow translational modifications such as glycosylation, phosphorylation, and fatty acylation. Related In-Fusion Cloning Products Kit Components (for 10 × 20-μL reactions) (1) Cell Lysate*1 100 μL (2) Mixture-1 60 μL (3) Mixture-2*2 10 μL (4) Mixture-3*2 20 μL (5) T7 RNA Polymerase (200 U/μL) 10 μL (6) pT7-IRES Vector (0.5 μg/μL) 20 μL (7) Control Vector*3 (0.3 μg/μL) 5 μL *1: Dissolve just prior to use; gently and thoroughly mix with a micropipette and use immediately. After use, promptly store at –80°C. Note: Although five cycles of freeze-thaw generally would not lead to any decline in performance, the cell lysate should be stored in aliquots of the required volume. *2: Mixture-2 and Mixture-3 contain protein. To avoid protein deactivation, do not stir excessively or vortex. Mixture-2 contains an HN-tagged protein. *3: This vector harbors a β-galactosidase gene.
4
His-Tag
For rapid and easy cloning, use the In-Fusion HD Cloning System (Clontech Cat. # 639645/ 639646/639692/ 639647) or In-Fusion HD Cloning System CE (Clontech Cat. # 639636/ 639637/639693/ 639638) to generate pT7-IRES constructs with your insert of interest.
Clontech, A Takara Bio Inc. Company • www.clontech.com/takara
2 Expression Systems
Figure 2. Principle of the Human Cell-Free Protein Expression System. A) In an easy and simple protocol, target protein translation is initiated by adding pT7-IRES Vector containing the target gene cassette and other kit components. B) The target gene RNA transcribed from the pT7-IRES Vector has an IRES sequence designed to promote protein translation initiation. As protein synthesis progresses, the translation initiation factor from the cell lysate becomes inactivated. The translation enhancement factor in the reaction mixture, however, reactivates this inactivated translation initiation factor and thereby maintains a high level of translation.
Relative absorbance
120
1
2
3
100 80 Mixture-2(＋) Mixture-2(ー)
60 40 20 0
0
1
2
3
4
5
6
7
8
◄◄
Time (hour)
Figure 3. Time course of β-galactosidase expression and effect of translation enhancement factor. Eight replicates of β-galactosidase in vitro translation were performed using 1 µL of Control Vector per reaction. At each of the indicated time points (0, 0.5, 1, 2, 3, 4.5, 6, and 8 h after start of the reaction), one reaction tube was removed and used for β-galactosidase activity assay with O-nitrophenyl-β-D-galactopyranoside (ONPG) as the substrate. A separate set of reactions were conducted in absence of the translation enhancement factor (Mixture-2). The activity of β-galactosidase increased over time to peak at approximately 4.5 h. Additionally, the presence of Mixture-2 containing translation enhancement factor markedly increased yield of active protein.
Figure 4. Synthesis of high molecular weight proteins using the Human Cell-Free Protein Expression System. In vitro translation reactions were performed to synthesize human Dicer (200 kDa, lane 2) or human eIF4G (170 kDa, lane 3) protein. Reactions were analyzed by SDS-PAGE and Coomassie blue staining. Arrowheads indicate target proteins. Lane 1, negative control.
Clontech, A Takara Bio Inc. Company • www.clontech.com/takara
5
Brevibacillus Expression System II
Expression Systems
2
Brevibacillus Expression System II Brevibacillus choshinensis Competent Cells pNC-HisE DNA pNC-HisF DNA pNC-HisT DNA pNCMO2 DNA pNY326-BLA DNA pNI DNA pNI-His DNA pNY326 DNA
HB200 HB116 HB123 HB122 HB121 HB112 HB114 HB131 HB132 HB111
1 Kit 100 µL x 10 10 µg 10 µg 10 µg 10 µg 1 µg 10 µg 10 µg 10 µg
Features • Efficient production of secreted or intracellular target proteins • Produces negligible amounts of extracellular protease – products remain intact in culture medium • Unlike E. coli, produces no endotoxins • Proteins are produced in active form • Easy to culture, handle, and sterilize
Description Brevibacillus choshinensis is a gram-positive bacterium with exceptional capacity for heterologous protein expression. The Brevibacillus Expression System II enables highly efficient production of target protein in secreted form. This system allows high yield of active proteins and is wellsuited for expression of eukaryotic proteins. The Brevibacillus system is nearly free of proteases, which facilitates production of intact protein products. Examples of successfully expressed proteins can be seen in Table 1. This includes expression of enzymes, antigens, and cytokines. Each protein was produced at a very high level of expression and confirmed to have native biological activity. In addition, proteins from taxonomically distant organisms were successfully produced, such as eubacteria, archaebacteria, eukaryotes, and viruses. The Brevibacillus system facilitates disulfide bond formation (commonly required in proteins of eukaryotic origin). In addition, B. choshinensis serves as an excellent host for intracellular protein production, frequently producing intracellular proteins in soluble form in the cytoplasm without forming inclusion bodies. The Brevibacillus system often works better than E. coli for expression of particular targets. Utilizing His-tag containing vectors (pNC-HisE, pNC-HisF, pNC-HisT, pNI-His) allows effective purification of the expressed target protein. Tags can be removed by protease treatment following purification.
6
Proteins Enzymes α-amylase α-amylase Sphingomyelinase Sphingomyelinase Xylanase Xylanase CGTase CGTase Chitosanase Chitosanase Hyper Hyperthermo-stable thermo-stableprotease protease Hyper Hyperthermo-stable thermo-stablenuclease nuclease PDI PDI Antigens Surface Surfaceantigen antigen Surface Surfaceantigen antigen Cytokines EGF EGF IL-2 IL-2 NGF NGF IFNIFN-γ γ TNF-α TNF-α GM-CSF GM-CSF GH GH
Origins
Origins
Production（ Production（ g/L ） g/L
B. licheniformis B. cereus B. halodurans B. macerans B. circulans A. pernix P. horikoshii human
3.7 3.0 3 0.2 1.5 1.4 0.1 0.7 1.0 1
E. rhusiopathiae T. pallidum
0.90.9 0.80.8
human human mouse chicken bovine bovine flounder
1.5 0.6 0.2 0.5 0.4 0.2 0.2
Table 1: Example of Proteins Expressed using the Brevibacillus Expression System System Components: Product Name Brevibacillus Expression System II Kit Components Expression Vectors pNY326A DNA pNCMO2 DNA Control Vector pNY326-BLA DNA Competent Cells Brevibacillus choschinesis Competent cells MT Medium Solu on A Solu on B
Clontech, A Takara Bio Inc. Company • www.clontech.com/takara
Catalog # HB200
Quan ty Kit
HB111 HB112
10 µg 10 µg
HB114
1 µg
HB116
100 µL x 10 tubes 1 ml x 10 1 ml 1 ml x 2
Vectors for the Brevibacillus System Lac operator P2 promoter sec signal peptide
pNC-HisE (5,263 bp)
pNC-HisF (5,260 bp)
rep
＋
ColE1 ori
(5.2 kb）
r
rep
ori +
2 rep
Ampr
P5 promoter sec signal peptide multiple cloning site X-terminator ori +
pNY326 (3.4 kp)
Nm
pNI DNA (5,055 bp)
rep
Nmr
r
multi cloning site
ColE1 ori
pNI-His DNA (5,079 bp)
rep
ori –
Lac operator
ori +
NmR
All shuttle vectors between B. choshinensis and E. coli contain the P2 promoter, which is one of the five promoters that control transcription of the cell wall protein gene (HWP). This promoter functions only in B. choshinensis and not in E. coli, thereby ensuring robust protein production only in B. choshinensis.
ori –
Ampr
NmR
ori
P2 promoter
rep
Nm r
Expression Systems
NmR
pNCMO2
pNC-HisT (5,260 bp)
Lac operator His-Tag multi cloning site
ColE1 ori
AmpR
AmpR
ori –
ori –
ori +
ColE1 ori
rep
P2 promoter
His-Tag MCS
ori –
ori＋
ColE1 ori
AmpR
Amp
His-Tag MCS
ori−
ori＋
ColE1 ori
Lac operator P2 promoter sec signal peptide
Lac operator P2 promoter sec signal peptide
His-Tag MCS
ori−
Nmr
The pNY326 vector* is maintained more stably than pNC or pNI vectors in the host cells due to much weaker promoter activity and smaller size (3.4 kb). The host strains containing the vector can be repeatedly subcultured, may be used for scaled-up production, and will continue to stably produce protein. The pNY326 vector must be constructed by a one-step method using B. choshinensis. Brevibacillus choshinensis Competent Cells are used as the transformation host. *Can only be maintained in B. choshinensis
Choosing a Brevibacillus Vector? His-Tag
Sec Signal Peptide
Construct in
Protease cleavage site
X-terminator
Yes
Yes
Yes
E. coli
Enterokinase
No
Secretory
Yes
Yes
Yes
E. coli
Thrombin
No
Shuttle Vector
Secretory
Yes
Yes
Yes
E. coli
Factor Xa
No
Shuttle Vector
Intracellular
Yes
Yes
No
E. coli
Enterokinase
No
Shuttle Vector
Intracellular
Yes
No
No
E. coli
No
No
Expression
Secretory
No
No
Yes
Brevibacillus
No
Yes
pNCMO2 (5.2 kb)
Shuttle Vector
Secretory
No
No
Yes
E. coli
No
Yes
pNY326-BLA
Positive Control
Secretory
Vector Name
Vector Type
pNC-HisE (5,263 bp)
Shuttle Vector
Secretory
pNC-HisT (5,260 bp)
Shuttle Vector
pNC-HisF (5,260 bp) pNI-His DNA (5,079 bp) pNI DNA (5,055 bp) pNY326 (3.4 Kb)
Expression Vector Lac Operator
Includes a gene encoding Bacillus licheniformis a-amylase (55 kDa)
Clontech offers a broad range of products for purifying His-tagged proteins. Please see the Clontech Related Products section on page (31) for ordering information.
Clontech, A Takara Bio Inc. Company • www.clontech.com/takara
7
B. subtilis Secretory Protein Expression System B. subtilis Secretory Protein Expression System
3380
10 rxns
Features
Description Bacillus subtilis has become an increasingly popular host for recombinant protein expression. With its ability to secrete protein directly into culture media, amenability to medium- and large-scale fermentation, lack of codon bias, and designation by the U.S. Food and Drug Administration as an organism that is Generally Regarded As Safe (GRAS), it’s no wonder that the majority of industrially-produced enzymes are expressed in Bacillus species such as B. subtilis. Optimization of secretion, however, can be necessary to achieve highest yields. To address this, the B. subtilis Secretory Protein Expression System from Takara Bio allows rapid development of a library of B. subtilis clones, each bearing a pBE-S construct in which the ORF for your protein of interest is fused with sequences for 173 unique signal peptides. Perform a downstream assay to identify and select clones which secrete the highest amount of functional protein into the culture media, and you can quickly identify the signal peptide that results in efficient expression of your desired secreted protein.
multi cloning site (MCS) His-Tag
pBE-S DNA (5,938 bp)
Kanr ColE1 Ampr
Figure 2. Vector map for pBE-S DNA, a B. subtilis/E. coli shuttle vector used with the B. subtilis Secretory Protein Expression System. Kit Components*1 SP DNA mixture (0.032 pmol/μL)*2 45 μL pBE-S DNA (0.5 μg/μL)*3 20 μg 100 μL x 2 B. subtilis RIK1285*4 (glycerol stock) *1: Library development (10 reactions) *2: DNA mixture encoding secretory signal peptides from the 173 types of B. subtilis for use with In-Fusion cloning system (10 reactions). For the sequence of secretory signal peptides, please see the product page for the B. subtilis Secretory Protein Expression System on the Takara Bio web site. *3: in TE buffer (pH 8.0) *4: Marburg 168 derivative: trpC2 , lys1 , aprE Δ3, nprR2 , nprE18 The System provides sufficient reagents for 10 library development reactions.
Storage • SP DNA mixture and pBE-S DNA: –20°C • B. subtilis RIK1285 glycerol stock: –80°C
A405
• Expression of soluble, recombinant protein secreted directly into the culture media • Protein expression in a host amenable to medium- and large-scale fermentation in addition to small-scale culturing • Expression of proteins with complex structure, such as proteins with disulfide (S-S) bonds • Generation of target protein in a host that is considered to be Generally Regarded As Safe (GRAS) by the U.S. Food and Drug Administration • Useful for producing easily purified recombinant protein – with proper in-frame cloning, a C-terminal His-tag can aid purification from culture media
52 I
SP
A405
Applications
promoter
pUB
A405
Expression Systems
2
173 different types of SP DNA are inserted into this region in place of the SP
I
• Includes pBE-S DNA, an E. coli/B. subtilis shuttle vector with B. subtilis-derived subtilisin (aprE) promoter, secretory signal peptide (aprE SP), Multiple Cloning Site, and 3’ (C-terminal) His-tag sequence • Supplied with SP DNA Mixture, a library of DNA sequences encoding 173 unique secretory signal peptides that can be inserted upstream of your target gene • Fully compatible with In-Fusion cloning kits and systems to allow rapid and easy construct generation • Includes B. subtilis strain RIK1285
3 2.5 2 1.5 1 0.5 0
1
3 2.5 2 1.5 1 0.5 0 161 3 2.5 2 1.5 1 0.5 0 321
11
21
31
41
51
61
71
81
91
101
111
121
131
141
151
◄
171
181
191
201
211
221
231
241
251
261
271
281
291
301
311
◄
331
341
351
361
371
381
391
401
411
421
431
441
451
461
◄
䠝㻮
Figure 3. Results of measuring β-glycosidase activity of 470 clones of an expression library with different signal peptide sequences. Clones showing activity levels of varying strengths were observed. The arrowheads indicate the expression level observed with the aprE signal peptide.
Related In-Fusion Cloning Products For rapid and easy cloning, use the In-Fusion HD Cloning System* (Clontech Cat. # 639645/639646/639692/639647) or In-Fusion HD Cloning System CE* (Clontech Cat. # 639636/639637/639693/639638) to generate pBE-S constructs with your insert of interest.
Figure 1. Flowchart of the experimental procedure for the B. subtilis Secretory Protein Expression System. 8
*: Available in the U.S. only. Outside of the U.S., use the In-Fusion HD Cloning Kit (Clontech Cat. # 639648/639649/639650) or In-Fusion HD Cloning Kit w/ Cloning Enhancer (Clontech Cat. # 639633/639634/639635) in combination with high-efficiency Stellar Competent Cells (Clontech Cat. # 636763/636766), a HST08 E. coli strain. Availability of In-Fusion systems and kits varies by geographic location; check for products sold in your region.
Clontech, A Takara Bio Inc. Company • www.clontech.com/takara
pCold Expression Vectors
pCold Vector Set pCold I DNA pCold II DNA pCold III DNA pCold IV DNA
3360 3361 3362 3363 3364
Features
1 Set (ea. 5 µg) 25 µg 25 µg 25 µg 25 µg
Description
Related Products
Takara’s pCold Expression Vectors offer Cold Shock expression technology for high purity, high yield protein production.
Chaperone Plasmid Set, 3340, p. 15.
The pCold series includes four different vectors. Each includes the Cold Shock Protein A (cspA) promoter for expression of highly pure recombinant protein in E. coli at high yield. These vectors selectively induce target protein synthesis at low temperature
Reference
• Great for difficult proteins that can’t be expressed with the T7 system • Facilitates increased solubility due to expression at reduced temperature • Facilitates increased purity due to repressed expression of host proteins
Application
1. Quing, G., et. al. (2004) Nature Biotechnol. 22(7):877-882.
Takara’s pCold expression vectors
pCold I
M
pCold II
cspA 3'UTR multiple cloning site cspA 5'UTR lac operator cspA promoter M
4.4kb
IG 13
pCold IV Amp
ColE1 ori
ColE1 ori
IG 13
pCold III Amp
4.4kb
Amp
Amp
4.4kb
IG 13
cspA 3'UTR multiple cloning site TEE cspA 5'UTR lac operator cspA promoter
lacI
M
lacI
IG 13
lacI
M
cspA 3'UTR multiple cloning site His•Tag TEE cspA 5'UTR lac operator cspA promoter
ColE1 ori
4.4kb
lacI
cspA 3'UTR multiple cloning site Factor Xa site His•Tag TEE cspA 5'UTR lac operator cspA promoter
ColE1 ori
In the following examples, genes that were poorly expressed or that produced insoluble protein with the T7 promoter expression system were expressed using the pCold system. pCold I DNA was used as an expression vector in E. coli. Expression from T7 promoter-driven vectors was induced with IPTG and T7 plasmid-containing cells were cultured at 37°C. N.C T7 pCold
T7
kDa
T
S
pCold T S
kDa
T: Entire protein fraction S: Soluble fraction
97.4 97.4
66.2
← Expression level increased
66.2
45 45
31
← Expression enabled
21.5
21.5
14.4
14.4
CBB staining of the entire protein fraction
Figure 1. Expression of human gene A.
31
Human gene A (~31 kDa) was expressed in both the T7 system and the cold-shock expression system. No expression was observed in the T7 system, but human gene A was expressed in the pCold system.
CBB staining
Figure 2. Expression of human gene C.
Comparison of expression of soluble human gene C protein (~80 kDa) in the cold-shock expression system vs. the T7 system was performed. Target protein in the soluble fraction of pCold cells was dramatically higher than that of the T7 system.
Clontech, A Takara Bio Inc. Company • www.clontech.com/takara
9
3 Products for Increased Protein Yield and Purity
• High-efficiency protein expression using a cold shock promoter
(15°C), a condition at which host protein synthesis is suppressed and protease activity is decreased. This results in high yields of target protein (~60% of intracellular protein). In addition to the cspA promoter, all four vectors contain a lac operator (for control of expression), ampicillin resistance gene (amp r ), ColE1 origin of replication, M13 IG fragment, and multiple cloning site (MCS). Three vectors also contain either a translation enhancing element (TEE), His-Tag sequence, and/or Factor Xa cleavage site. These vectors work equally well for synthesis of non-labeled and radiolabeled proteins and can be used with Takara’s Chaperone Plasmid Set (Cat. # 3340).
pCold TF Vector pCold TF DNA
3365
25 µg
Application
3
• Highly efficient protein expression using cold shock technology • High yield of active protein due to Trigger Factor chaperone as a solubility-promoting fusion tag
kDa
pCold 1 2
pCold + Chaperone 1 2
T7 1 2 1. Cell extract solution 2. Soluble fraction target protein
97
Description
Products for Increased Protein Yield and Purity
pCold TF 1 2
66
Takara’s pCold TF DNA Vector is a fusion cold shock expression vector that expresses Trigger Factor (TF) chaperone as a soluble fusion tag. Trigger Factor is a 45 kDa prokaryotic ribosome-associated chaperone protein that facilitates co-translational folding of nascent polypeptides. Because of its E. coli origin, TF is highly expressed in E. coli expression systems. The pCold TF DNA Vector consists of the cspA promoter plus additional downstream sequences including a 5’ untranslated region (5’ UTR), a translation enhancing element (TEE), a His-Tag sequence, and a multiple cloning site (MCS). A lac operator is inserted downstream of the cspA promoter to ensure strict regulation of expression. Additionally, recognition sites for HRV 3C Protease, Thrombin, and Factor Xa are between TF-Taq and the Multiple Cloning Site (MCS). These sequences facilitate tag removal from the expressed fusion protein. Most E. coli strains can serve as expression hosts.
45
* co-expressed trigger factor
*
31 22
Figure 1. Expression of protein A in T7 and pCold systems
The expression of enzyme protein A (~29 kDa) was poor when utilizing aT7 or pCold I expression systems, even when the pCold I construct was co-expressed with a chaperone. In contrast, the expression of target protein as a fusion (29 kDa + 52 kDa) was successful with pCold TF DNA, and most of the expressed protein was in soluble form. The expressed enzyme protein A showed activity even in the form of a fusion protein (data not shown).
pCold ProS2 DNA pCold™ ProS2 DNA
3371
25 µg
Features
Description
• Facilitates high yield protein expression with optimized protein folding • Enables expression of fusion proteins with a soluble tag to optimize solubility
The pCold Pro S2 expression vector features Protein S, a soluble tag from Myxococcus xanthus fused to the N-terminus of target proteins. Tight regulation of protein expression is maintained by a lac operator downstream of the cold shock promoter. HRV 3C Protease, Thrombin, and Factor Xa recognition sites are encoded between the Protein S tag and the MSC to facilitate tag removal.
mRNA Interferase™-MazF Enzyme mRNA Interferase™-MazF
2415A
1000 units
Application
Description
• Site-dependent cleavage of ssRNA
MazF is a toxin protein in the toxin-antitoxin module of E. coli. It possesses endoribonuclease activity and specifically cleaves single-stranded RNA at the 5’ end of 5’-ACA-3’ sequences. This enzyme does not cleave double-stranded RNA, doublestranded DNA, or single-stranded DNA. mRNA Interferase-MazF is supplied as a fusion protein of E. coli MazF and Trigger Factor. The Trigger Factor protein is an E. coli chaperone protein. The enzyme is also supplied with a 5X MazF buffer (200 mM Sodium phosphate, pH 7.5, 0.05% Tween 20.)
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Clontech, A Takara Bio Inc. Company • www.clontech.com/takara
SPP System SPP System™ I SPP System™ II SPP System™ III SPP System™ IV SPP System™ I-IV
3367 3368 3369 3370 3366
1 kit 1 kit 1 kit 1 kit 1 kit
3
Application • P referential expression of target protein by suppression of endogenous proteins using mRNA interferase plasmid
Products for Increased Protein Yield and Purity
Description This system utilizes an E. coli protein, MazF, described as an mRNA interferase by Suzuki et. al. MazF is a sequence-specific endoribonuclease that cleaves single strand RNAs at 5’-ACA-3’ (ACA) sequences. When using the SPP System, the transcript of interest should therefore lack ACA sequences. MazF is co-expressed in the E. coli host and suppresses expression of non-target genes by cleaving host transcripts at ACA sequences. Therefore, the target protein is the most abundantly expressed protein (Figure 1). Because of the requirement for target gene transcripts to lack ACA sequences, the SPP System is not suitable for all genes of interest; however, when appropriate, it can result in extremely high levels of protein production.
Reference Suzuki, M., et. al. (2005) Molecular Cell 18(2)253-261.
Figure 1. Synthesis of cspA-promoter expressed envAB in presence and absence of MazF.
E. coli BL21 cells co-expressing MazF and pCold (SP-4) envZB showed good envZB expression and extremely low background synthesis of host proteins.
Clontech, A Takara Bio Inc. Company • www.clontech.com/takara
11
pBApo-CMV Vectors pBApo-CMV Neo DNA pBApo-CMV Pur DNA pBApo-CMV DNA
20 µg 20 µg 20 µg
Features • General purpose gene expression vector for mammalian cells • Can be used to express miRNA precursors and other transcription products • Allows easy transfer of expression cassette to adenovirus vector
EcoR V Cla I
EcoR V
EcoR I
SV40 polyA
BamH I Xba I
Description
Sse8387 I
HSV TK polyA EcoR V
pBApo-CMV is a general purpose gene expression vector for mammalian cells. This vector has a promoter from cytomegalovirus (CMV IE promoter), a poly(A) signal from thymidine kinase of herpes simplex virus (HSV), and a multiple cloning site (MCS). This vector can be used to express miRNA precursors and other transcription products in addition to protein-coding genes. The cassette promoter + ORF + poly(A) signal cassette can be easily transferred from this vector to an adenovirus vector.
Sph I
HSV TK polyA
Cla I
or
BamH I Xba I Sal I Acc I Hinc II Pst I/Sse8387 I
CMV IE promoter
Hind III
pBApo-CMV Neo
EcoR I
Cla I
CMV IE promoter
NeoR QT PurR
Hind III EcoR V
pBApo-CMV Pur
Cla I
pBApo-CMV
SV40 promoter
AmpR
AmpR
With the ability to achieve a high infection efficiency across a broad spectrum of cell lines, adenovirus vectors are suitable for in vitro and in vivo gene transduction. For constructing recombinant adenoviruses, use Adenovirus Expression Vector Kit (Dual Version) Ver. 2 (Cat. # 6170). In addition to a basic vector (Cat. # 3242), the pBApo-CMV series also includes vectors with a neomycin resistance cassette (Cat. # 3240) or a puromycin resistance cassette (Cat. # 3241) for stable expression in mammalian cells.
Figure 1: Expression of DsRed-Express with pBApo-CMV Neo. HEK 293 cells were transfected with a pBApo-CMV Neo construct bearing a DsRed-Express cassette. The cells were visualized by fluorescence microscopy 2 days after transfection.
pBApo-EF1alpha pBApo-EF1alpha Neo DNA pBApo-EF1alpha Pur DNA
3243 3244
20 µg 20 µg
Features
RV
RV I
• EF1 alpha promoter directs higher level of expression than CMV IE promoter • Can be used to express miRNA precursors and other transcription products • Allows easy transfer of expression cassette to adenovirus vector
Application
I
RI
HI
RI
EF1α promoter
SV40 polyA
EF1α promoter
SV40 polyA
I
NeoR
HI 8387 I
I
PurR
d III
d III
RV
RV I
pBApo-EF1α Neo 5,167 bp
SV40 promoter
8387 I
HSV TK polyA
HSV TK polyA
pBApo-EF1α Pur
I
4,972 bp
SV40 promoter
• Gene expression in mammalian cells AmpR
AmpR
Description Transient expression %positive 70
300
60
250
20
60
35
50
EFp CMVp
150
EFp CMVp
100
25 20 15
40 EFp CMVp
MFI
200
% positive
30
MFI
40
30
50 40
Stable Expression %positive
MFI
MFI
The pBApo-EF1alpha series includes simple gene expression vectors for mammalian cells. These vectors include a promoter from human polypeptide chain elongation factor (EF-1 alpha promoter) and a poly(A) signal site from herpes simplex virus thymidine kinase gene. They can also be used to express miRNA precursors and other transcripts in addition to protein-encoding genes. Versions with neomycin or puromycin resistance cassettes are available. A promoter + ORF + poly(A) signal cassette can be easily removed from these vectors and transferred to an adenovirus vector. Adenovirus vectors, which have high infection efficiency and a wide target cell spectrum, are suitable for in vitro and in vivo gene transductions. For constructing recombinant adenoviruses, the Adenovirus Expression Vector Kit (Dual Version) Ver. 2 (Cat. # 6170) is recommended.
% positive
Mammalian Expression Vectors
4
3240 3241 3242
30
EFp CMVp
20
10 10
10
50
0
0
0
0
ES-E14TG2a
ES-E14TG2a
ES-E14TG2a
ES-E14TG2a
5
Figure 2: Expression levels of pBApo-EF1α Neo or pBApo-CMV Neo. Expression of AcGFP1 was assessed in mouse ES cells transiently or stably expressing the target gene under the direction of either the EF1α promoter or CMV IE promoter.
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Clontech, A Takara Bio Inc. Company • www.clontech.com/takara
pDON-AI-2 pDON-AI-2 DNA pDON-AI-2 Neo DNA
3654 3653
20 µg 20 µg
Features
5' LTR
Amp
HCMV IE promoter MLV R MLV U5
r
PmaCI (1261) Apa I (1266) Sac II (1270) Not I (1272) Bgl II (1280) Cla I (1286) Bam HI (1292) Sal I (1298) Hpa I (1304)
SD
ψ
pDON-AI-2 Intron + SA
4586 bp
MLV U3
Amp
HCMV IE promoter MLV R MLV U5
r
PmaCI (1261) Apa I (1266) Sac II (1270) Not I (1272) Bgl II (1280) Cla I (1286) Bam HI (1292) Sal I (1298) Hpa I (1304)
SD
ψ
Intron + SA
pDON-AI-2 Neo
Minimal SV40 promoter
5719 bp r
Neo
MLV R MLV U5
MLV U5 MLV U3 MLV R
3' LTR
Application
3' LTR
• Retrovirus-mediated gene transfer into mammalian cells
Description The retroviral vector pDON-AI-2 and pDON-AI-2-Neo do not contain any MoMLV derived genes (gag, pol, or env coding sequences) except LTR and packaging signal (psi sequence). The U3 region of 5’ LTR has been substituted with a stronger promoter derived from cytomegalovirus, giving these vectors a high transcription efficiency
and allowing them to be used to generate high titer-recombinant retroviruses and accordingly efficient gene transductions. Moreover, they carry a human actin-derived intron and splice acceptor upstream of the cloning site to increase the efficiency of target gene expression after gene transduction. pDON-AI-2 Neo DNA (Cat.# 3653) has a neomycin resistance gene as a drug selection marker.
pMEI-5 pMEI-5 DNA pMEI-5 Neo DNA
3656 3655
20 µg 20 µg
Features • For highly efficient transcription • To increase target gene expression after gene transduction, includes a human EF1αderived intron with high splicing activity upstream of the cloning site
Application
Amp
Amp
r
MLV U3
MLV U5
pMEI-5
The retroviral vectors pMEI-5 and pMEI-5-Neo possess LTR and psi (viral packaging signal), but lack the structural genes necessary for particle formation and replication (gag, pol, and env). Because these vectors contain a human EF1α-derived intron with high splicing capacity, they facilitate high transcription efficiency. pMEI-5 Neo DNA
SD
ψ
4689 bp
MLV U5 MLV U3 MLV R
3' LTR
Description
5' LTR MLV U3 MLV R MLV U5
MLV R
Intron + SA
• Retrovirus-mediated gene transfer into mammalian cells
r
5' LTR
MCS PmaCI (1760) Apa I (1765) Sac II (1769) Not I (1771) Bgl II (1779) Cla I (1785) Bam HI (1791) Sal I (1797) Hpa I (1803) Xho I (1811)
5820 bp
MLV U5 MLV R MLV U3
3' LTR
Intron + SA
minimal SV40 promoter
Neo
MCS SD
ψ
pMEI-5 Neo
PmaCI (1760) Apa I (1765) Sac II (1769) Not I (1771) Bgl II (1779) Cla I (1785) Bam HI (1791) Sal I (1797) Hpa I (1803)
r
contains the neomycin resistance gene as a selective marker. Gene expression levels 2- to 8-fold higher than pDON-Al-2 series can be expected.
Clontech, A Takara Bio Inc. Company • www.clontech.com/takara
4
13
Mammalian Expression Vectors
• Enables highly efficient gene transduction • Retroviral vector that lacks all MoMLV-derived genes (gag , pol , or env) except LTR and packaging signal (Ψ sequence) • Includes a human actin-derived intron and splice acceptor for efficient target gene expression
MCS
5' LTR
MCS
pDON-5 pDON-5 DNA pDON-5 Neo DNA
4
3658 3657
20 µg 20 µg 5' LTR
• High gene transduction and high transcription efficiency • Enables the production of high titer recombinant retroviruses
Amp
r
SD
ψ
pDON-5 Intron + SA
4697 bp
HCMV IE promoter MLV R
MCS
HCMV IE promoter MLV R MLV U5
Application
MLV U3
PmaCI (1370) Apa I (1375) Sac II (1379) Not I (1381) Bgl II (1389) Cla I (1395) Bam HI (1401) Sal I (1407) Hpa I (1413) Xho I (1421)
Amp
r
MLV U5
PmaCI (1370) Apa I (1375) Sac II (1379) Not I (1381) Bgl II (1389) Cla I (1395) Bam HI (1401) Sal I (1407) Hpa I (1413)
SD MLV U5
ψ
Intron + SA
pDON-5 Neo 5828 bp
Minimal SV40 promoter r
Neo MLV U5 MLV U3 MLV R
MLV R
• Retrovirus-mediated gene transfer into mammalian cells
3' LTR
3' LTR
Description The pDON-5 and pDON-5 Neo vectors facilitate both high-efficiency gene transduction and high expression. They allow higher production of high-titer recombinant retroviruses than pMEI-5 or pMEI-5-Neo (see Figure 1). By transfecting the vector pDON-5 or pDON-5 Neo vector into an appropriate packaging cell line, the vector expresses transient or stable transcribed product containing virus packaging signal (psi) and target gene and selective marker.
ivp/ml
Mammalian Expression Vectors
MCS
5' LTR
Features
8,000,000
3.5
7,000,000
3.0
6,000,000
2.5
5,000,000
2.0
4,000,000
1.5
3,000,000
1.0
2,000,000
0.5
1,000,000
0
0 DON-AI-2 DON-AI-2 MEI-5 Neo Neo
MEI-5
DON-5 Neo
DON-5
Figure 1: Comparison of Virus Titers (ZsGreen/HT1080) The ZsGreen gene was inserted into the Bam H I/Hpa I site of the indicated vectors. G3T-hi cells were transiently transfected and then 2 sets of recombinant retroviruses were produced for each viral vector.
14
These vectors possess LTR and psi (viral packaging signal), but not the structural genes necessary for particle formation and replication (gag, pol, and env). These vectors include a strong cytomegalovirus promoter (HCMV IE) within the U3 region of 5’ LTR and a human EF1α-derived intron with high splicing activity upstream of the multiple cloning site. pDON-5 Neo includes the neomycin resistant cassette as a selective marker.
DON-AI-2 Neo
DON-AI-2
MEI-5 Neo
MEI-5
DON-5 Neo
DON-5
Figure 2: ZsGreen Expression Intensity (relative value to the mean fluorescence intensity of 1 copy/cell) HT1080 cells were infected with recombinant retroviruses at various dilution rates using polybrene. Three days after transduction, gene transfer efficiency and ZsGreen expression intensity were measured using a flow cytometer. Values shown were normalized using the expression intensity of DON-AI-2 virus as 1.
Clontech, A Takara Bio Inc. Company • www.clontech.com/takara
Chaperone Plasmid Set Chaperone Plasmid Set
3340
1 Set
Application araC
• Promotes correct in vivo folding of expressed recombinant proteins in E. coli
groEL
araB dnaK
Description
pG-KJE8 11.1 kb
araC
groES Pzt1
araB
Pzt1 araB
gro ES pACYC ori pACYC ori
Cmr
pKJE7
dnaK
groEL
7.2 kb
araC
pG-Tf2
pTf16
8.3 kb
grpE dnaJ
tetR
tig
gro EL
Cmr
5 kb
tig pACYC ori
Resistance Plasmid pG-KJE8 pGro7 pKJE7 pG-Tf2 pTf16
Chaperone Promoter Inducer Marker dnaK-dnaJ-grpE-groES-groEL araB, Pzt1 L-Arabinose, Tetracycline Cmr groES-groEL araB L-Arabinose Cmr dnaK-dnaJ-grpE araB L-Arabinose Cmr groES-groEL-tig Pzt1 Tetracycline Cmr tig araB L-Arabinose Cmr
Plasmids with the Chaperone Plasmid Set work well in combination with the pCold expression system vectors.
References
Kit Components
2. Nishihara, K., et al. (1998) Appl. Environ. Microbiol. 64(5):1694-1699.
1. Nishihara, K., et al. (2000) Microbiol. 66(3):884-889.
5 plasmids: conc. 10 ng/µL; 100 µL each plasmid
Human gene A (~70 kDa) was expressed in insoluble form when using pCold I alone. However, the level of soluble expressed protein increased significantly when the chaperone plasmid pG-Tf2 was co-expressed with the pCold I construct.
Human gene B (~24 kDa) was not expressed when using pCold I DNA alone. However, co-expression with the chaperone plasmid pG-Tf2 resulted in expression of high levels of target protein in soluble form.
The combination of Cold Shock Expression Vectors and the Chaperone Plasmid Set often leads to significant improvement in expression level of soluble forms of target proteins. If sufficient expression or solubilization cannot be achieved using pCold vectors alone, we recommend co-expression with chaperone plasmids. Furthermore, pCold vectorbased expression systems may produce better results by co-expressing chaperone plasmids carrying the tig sequence, such as pG-Tf2 or pTf16, which are included in the Chaperone Plasmid Set (data not shown).
Clontech, A Takara Bio Inc. Company • www.clontech.com/takara
15
Folding
Note that this system cannot be used in combination with chloramphenicol-resistant E. coli host strains or expression plasmids that carry a chloramphenicol-resistance gene. For example, E. coli BL21(DE3), which is often used with pET systems, is a compatible host strain. However, E. coli BL21(DE3) pLysS and BL21(DE3) pLysE, which contain pLysS or pLysE plasmids that have the pACYC replication origin and the Cmr gene, cannot be used with this system.
groES
rrnBT1T2
araC
5
araB
pACYC ori grpE
Cmr
pACYC ori
pGro7 5.4 kb
tetR dnaJ
The Chaperone Plasmid Set consists of 5 different plasmids, each of which is designed to express multiple molecular chaperones. Together, they function as a “chaperone team” to facilitate protein folding. Co-expression of a target protein with one of these plasmids increases the recovery of soluble proteins. Each plasmid carries an origin of replication derived from pACYC and a Cmr gene, which allows use with E. coli expression systems utilizing ColE1-type plasmids with an ampicillin resistance gene as a marker. The chaperone genes are situated downstream of an araB or Pzt-1 (tet) promoter. Therefore, expression of target proteins and chaperones can be induced individually if the target gene is placed under the control of other promoters (e.g. lac). These plasmids also contain the necessary regulator (araC or tetR) for each promoter.
Cmr
Cmr
Chaperonin GroE Chaperonin Gro EL Chaperonin Gro ES
Folding
5
7330 7331
5 mg 0.5 mg
Application
Description
• Facilitates refolding of denatured proteins
Chaperonin GroE is a protein complex composed of GroEL (14 subunits, 57 kDa) and GroES (7 subunits, 10 kDa). It is thought to support the ability of proteins to form tertiary structure upon or immediately after translation. GroE is essential to assembly (and presumably reassembly after denaturation) of protein complexes in vivo. Chaperonin GroEL and GroES can be used for refolding denatured proteins to recover functional activity.
Corystein™ (Purothionin) Reagent Corystein™ (Purothionin) Reagent
7311
5 mg
Application
Description
• Facilitates protein refolding by promoting exchange reactions between disulfide bonds
Corystein™ (Purothionin) Reagent is a polypeptide purified from wheat endosperm. It catalyzes the formation of correct disulfide bonds in proteins. Corystein™ Reagent can be used alone or together with thioredoxin on a variety of proteins to re-form disulfide bonds.
Refolding CA Kit Refolding CA Kit Refolding CA Kit
7350 (small) 7351 (large)
Application
25 reactions 1 kit
Kit Components
• Refolding of isolated inclusion body proteins
Description The Refolding CA Kit uses a novel artificial chaperone technology (licensed from NFRI, BTRAI, and Ezaki Glico Co, Ltd.) in an easy 2-step procedure for optimizing the refolding conditions of inclusion body proteins. Optimization allows identification of the best conditions for correct protein folding and restoration of protein activity. The Small Kit (Cat. #7350) is supplied with guanidine hydrochloride and DTT for protein denaturation, four different surfactants that can be added independently to the unfolded protein solution to protect against molecular aggregation, and highly polymerized cycloamylose (CA), an artificial chaperone, for surfactant removal and recovery of protein activity. Overnight incubation of the CA-treated protein is followed by a quick 10-minute centrifugation. The resulting supernatant contains the refolded protein. The Large Kit (Cat. #7351) is used for large-scale refolding after initial determination of with the Small Refolding CA kit, and consists only of denaturant and CA.
7350 (Small Kit) 8 M guanidine hydrochloride (GdmCl) 4 M dithiothreitol (DTT) 4 surfactants: 1% Tween 40 1% Tween 60 1% CTAB (cetyltrimethylammoniumbromide) 1% SB3-14 (myristylsulfobetaine) 200 mM DL-cystine 3% CA (highly polymerized cycloamylose)
2 x 1 mL 50 µL 2 x 1 mL 2 x 1 mL 2 x 1 mL 2 x 1 mL 2 x 0.75 mL 7 x 1.6 mL
7351 (Large Kit) 3% CA (highly polymerized cycloamylose) 8 M guanidine hydrochloride (GdmCl)
6 x 20 mL 2 x 10 mL
References
Related Products
1. Machida, S., et al. (2000) FEBS Lett. 486(2):131-135.
Chaperone Plasmid Set, 3340, p. 15.
2. Sundari, C.S., et al. (1999) FEBS Lett. 443(2):215-219. 3. Daugherty, D.L., et al. (1988) J. Biol. Chem 273(51):33961-33971.
Principle of the Refolding CA Kit
Guanidi ne hydrochloride unfolds inclusion bodies
16
Surfactants prevent protein aggregation
Highly polymer ized CA removes surfactants and facilitates otein pr refolding
Clontech, A Takara Bio Inc. Company • www.clontech.com/takara
Biolo g icall y active protein in thermodynamically stable native conformati on
High-level Secretion of Recombinant Protein using the Brevibacillus Expression System By Michikazu Tanio and Toshiyuki Kohno, Mitsubishi Kagaku Institute of Life Sciences (MITILS), Tokyo, Japan
Analysis of Expression The obtained colonies were cultured for 1-3 days with 3-5 ml of media in a 14-18 mm diameter tube (37°C, 200 rpm reciprocal shaking culture). Two kinds of media were prepared: 2SYNm (2% glucose and 4% soytone and 0.5% yeast extract and 1 mM calcium chloride and 50 μg/ml neomycin) and TMNm (1% glucose and 1% polypeptone and 0.5% bonito extract and 0.2% yeast extract and 0.001% iron sulfate and 0.001% manganese sulfate, 0.0001% zinc sulfate, and 50 μg/ml neomycin). Expression analysis by SDS-PAGE and western blot with anti-His antibody showed that FD1 could be detected only in 2SYNm medium and FKBP was produced in both 2SYNm and TMNm media. 2SYNm medium was selected for large-scale culture due to its ease of preparation.
The yeast protein expression system using Pichia pastoris is known to be quite efficient for expression of proteins containing S-S bonds, but expression levels are highly variable among target proteins.
Large-Scale Culture and Purification
Various types of proteins have been successfully expressed using insect cells or animal cells, including target proteins that could not be expressed with E. coli systems, but E. coli remains superior for productivity and cost. However, a major advantage of eukaryotic expression systems including yeast is the capacity for post-translational modification such as phosphorylation or glycosylation, although such modifications would be an obstacle in structural analysis by leading to protein heterogeneity. Cell-free expression systems can sometimes express cytotoxic proteins, and avoid metabolic issues when performing selective amino-acid labeling with stable isotopes. However, cell-free reactions are performed in a reductive environment, thus preventing expression of proteins containing disulfide bonds. These systems are also of relatively high cost. Thus, each heterologous protein expression system has advantages and drawbacks and selection of an appropriate system must consider both cost and research purpose. However, there is no satisfactory system suitable for proteins containing disulfide bonds. In our laboratory, we have analyzed protein structure and function using different expression systems. The Brevibacillus Expression System was introduced recently. This expression system, which Takara Bio launched in 2006, has already shown numerous successes for producing secreted recombinant proteins (1-4). With this system we could obtain some secreted or cytoplasmic proteins that failed to be expressed with an E. coli system or a yeast system (5-7). Considering the simple protocol and affordable cost of the Brevibacillus Expression System, it should be a first choice - before the E. coli expression system - for recombinant protein expression.
1 ml of bacterial pre-culture incubated in 3-5 ml of 2SYNm medium was added to a 500 ml flask with 100 ml of 2SYNm or SYNm medium (2% glucose and 0.8% soytone and 0.5% yeast extract and 50 µg/ml neomycin) and incubated at 27°C or 37°C at 100 rpm for 1-5 days. The number of culture flasks was increased as needed according to the desired amount of protein expression and the end use of recombinant target protein. The expressed proteins were purified by affinity chromatography with TALON® Metal Affinity Resin (Clontech) after adjusting the culture supernatant pH to 8. FD1 was subjected to ion exchange and gel filtration chromatography (Fig 1), and FKBP was purified by gel filtration chromatography only.
Stable Isotopic Labeling 1 ml of the bacterial pre-culture in 2SYNm was added into 500ml flask with 100 ml of Stable Isotope Labeling C.H.L. Medium (Chlorella Industry Co., Ltd.) added by 50 μg/ ml neomycin and incubated at 27-37°C, 100 rpm for 1-5 days. The labeled proteins were purified by affinity chromatography and gel filtration chromatography. For amino acid-selective labeling, the unlabeled C.H.L. medium added by labeled amino acids (100 mg/L) was used as culture media.
In this paper, we describe both recombinant protein production and stable isotope labeling using the Brevibacillus Expression System.
Methods Expression and Purification of Two Recombinant Proteins - Human M-Ficolin Recognition Domain (FD1: Molecular weight 26.8 kDa) and Human FK506 Binding Protein (FKBP: Molecular weight 13 kDa)
Construct Design After amplification by PCR using Human Universal QUICK-Clone™ cDNA II (Clontech) as a template, each cDNA encoding the target protein was cloned into the pNCMO2 vector with insertion of a C-terminal 6 x His-Tag. The expression vector constructs were transformed into Brevibacillus choshinensis Electro-Cells with the electroporation protocol recommended by the manufacturer's user manual.
Figure 1. Expression and purificaton of FD1 protein with the Brevibacillus Expression Sytem. Lane 1: Culture supernatant after 2 days. Lane 2: FD1 protein purified by Talon. Lane 3: FD1 protein purified by ionic exchange and gel filtration chromatography.
Clontech, A Takara Bio Inc. Company • www.clontech.com/takara
17
6 Application Notes
Introduction A variety of currently used heterologous protein expression systems has been developed, such as E. coli, yeast, insect cells, animal cells and cell-free systems. Such expression systems enable the production of proteins that are difficult to isolate from raw material, and allow introduction of mutations, heavy atoms, and isotopes. The most popular and first choice in most cases is the E. coli protein expression system, which features high productivity, ease of use, and relatively low cost. However, many proteins are often produced in E. coli systems in insoluble inclusion bodies, particularly for proteins with disulfide(S-S) bonds, secreted proteins, or proteins originating from higher organisms. Refolding of inclusion bodies or of target proteins secreted to the periplasm can be performed, but this generally leads to lower production yields of active protein.
Results
Stable Isotope Labeling
Secretion by the Brevibacillus Expression System
Application Notes
6
FD1, which contains two S-S bonds and includes a substrate binding domain of human M-Ficoline, functions as a foreign substance recognition protein during innate immunity (Fig 2). Although we have already reported the FD1 crystallographic structure by secretion of FD1 with the yeast expression system P. pastoris (8-10), at the time of the original studies we could hardly produce any FD1 derivatives in this yeast expression system. With the Brevibacillus Expression System, however, we succeeded in the production of secreted FD1 protein and several FD1 derivatives with normal levels of activity (6). With regard to expression conditions, we found most efficient production occurring at 200 rpm for a shaking culture in test tubes and at 100 rpm for rotary shaking cultures in flasks. We observed lower production at higher rpm levels. The most appropriate culture temperature was 37°C. However, in one case of a certain FD1 variant, the protein expressed at 37°C showed no substrate binding activity, even though normal binding activity occurred upon culture at 27°C. This implies that, especially for the derivatives, irreversible degeneration might occur in the stable protein, even though the target protein has been secreted efficiently. Several experiments with different media compositions showed that media containing ~0.8 % soytone without calcium chloride is the most suitable for production of secreted proteins. A similar trend occurred not only for FD1 but also FKBP. On the other hand, pre-culture from a single colony with SYNm media Figure 2. Crystal structure of FD1 protein under this condition resulted (PDB ID 2D39) (9). in notably low growth. Taking all results into consideration, we chose our final culture condition for pre-culture as 3-5 ml of 2SYNm medium at 37°C with shaking (200 rpm) overnight (15-18 h), and for large-scale culture as 100 ml of SYNm medium at 27°C with rotary shaking (100 rpm) for a few days (2-4 days). Under these conditions, we could obtain ~10 mg of purified FD1 protein from 1 L culture, slightly higher than we obtained using the yeast expression system (5-8 mg / L). With activity analysis of FD1 derivative proteins obtained using the optimized culture conditions described above for the Brevibacillus Expression System, we have successfully identified the residues involved in FD1 substrate binding (6). In other studies, FKBP, a cytoplasmic protein lacking S-S bonds, was secreted with the Brevibacillus Expression System (5, 7). The culture and purification conditions were almost the same as described above for FD1 above except that the temperature for largescale culture was 37°C instead of 27°C because of the higher protein stability for FKBP. The yield of the purified FKBP protein with 2SYNm medium was about 16 mg/L. In our laboratory, we have successfully used the Brevibacillus Expression System to produce other cytoplasmic proteins in addition to FKBP. One protein of E. coli origin, which appeared as a mixture of monomer and dimer due to the S-S bond connecting one protein molecule to another in the oxidized environment of the medium, could be obtained as a homogenous population of monomers with this expression system by adding 10 mM of dithiothreitol (DTT) during the purification steps. In another case with a protein of human origin, expression occurred in the cytoplasm of E. coli, but the E. coli-expressed protein was degraded during the extraction process possibly because of endogenous E. coli protease activity. With the Brevibacillus Expression System, however, this protein was successfully secreted without degradation. These results suggest that the Brevibacillus Expression System is useful not only for secretory proteins with S-S bonds but also for the expression of cytoplasmic proteins. 18
Methods for stable isotope labeling with the Brevibacillus Expression System had not been established prior to the studies summarized here (5-7). Stable isotope labeling of a target protein is an indispensable technique for protein structural and functional analyses, especially for NMR studies. We tried to establish the stable isotope labeling with Brevibacillus Expression System, motivated to do so by the necessity of NMR analysis of FD1. We used FKBP as a model protein because the NMR signals for that protein were already assigned and because FKBP contains all 20 types of amino acids. After performing protein expression tests with several bacterial minimal media, including M9 minimal medium, Stable Isotope Probing C.H.L. Medium, manufactured by Chlorella Industry Co., LTD., Japan, showed the best results in terms of both growth and of expression. The protein yield of purified FKBP in C.H.L. medium was about 2-13 mg/L culture (at 37°C for 1-2 days). Considering that 1) about 91% of FKBP amino acids were labeled with 15N when using 15N Labeling C.H.L. medium, and 2) the 1H-15N HSQC NMR spectrum was quite similar to the data obtained with E. coli expression systems, we could confirm that the purified proteins expressed with the Brevibacillus Expression System maintained their normal folding structure. In addition, we tested special media in which each individual kind of 15N labeled amino acid was added to the non-labeled C.H.L. medium, trying a total of 19 kinds of amino acids except proline. As a result of 1H-15N HSQC NMR spectrum analysis on the labeled protein obtained with these 19 kinds of C.H.L. medium containing each 15N labeled amino acid, we found that selective labeling is possible with the Brevibacillus Expression System for nine kinds of amino acid residues (Table 1). On the other hand, the acidic and aromatic amino acids are metabolized to other amino acids from the first day of culture. Glycine, isoleucine, leucine, serine or threonine are each metabolized to other amino acids, with the end products being specific to each residue. We decided to harvest cysteine-labeled FKBP after 1 day of culture because FKBP was observed to degrade after 2 days culture. Such degradation was also observed after 3 days culture in the tyrosine-labeled C.H.L medium. Next, in order to show the result of stable isotopic labeling of FD1, we tried to perform the experiment in a similar way as described for FKBP labeling. We found that with the simple C.H.L. medium only, the FD1 yield was not as good, but we obtained better yield with the special C.H.L. medium (C.H.L.aa) where 100 mg/L of eight kinds of amino Amino Acids
Metabolism
Metabolism Rate (%)**
Selection
Ala
-
< 20
++
Arg
-
< 35
++
Asn
-
< 30
++
Asp
almost all aa
-
-
Csy
-
< 18
++
Gln
-
< 61
++
Glu
almost all aa
-
-
Gly
Cys, Ser, Trp
-
+
His
-
< 25
++
Ile
Leu, Val
-
+
Leu
Ile, Val
-
+
Lys
-
< 26
++
Met
-
< 16
++
Phe
almost all aa
-
-
Ser
Cys, Gly, Trp
-
+
Thr
Cys, Gly, Ser, Trp
-
+
Trp
almost all aa
-
-
Tyr
almost all aa
-
-
Val
-
< 23
++
Table 1. Amino acid selectivity of Brevibacillus* (7). Brevibacillus choshinensis HPD31-SP3. **Estimated by comparing average NMR signals obtained by 1H 15N HSQC NMR spectrum results of 15N labeled FKBP proteins.
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acids (without cysteine), respectively, were utilized out of the nine residues that can be labeled selectively with the Brevibacillus Expression System. Therefore, for the purpose of producing selectively-labeled FD1 protein, we decided to use the C.H.L.aa medium where a target amino acid is labeled with stable isotope, or the C.H.L.aa medium with the labeled cysteine. This improvement has made the amino acid selective labeling of FD1 possible, and we have successfully obtained several 1H-15N HSQC NMR spectra on FD1 (Fig 3A-C). The protein yield of purified FD1 was 2-5.5 mg/L (after 5 days of culture). Moreover, degradation of cysteine-labeled target protein was not observed in FD1. In conclusion, we have established a simple and cost-effective Stable Isotope Labeling method with the Brevibacillus Expression System.
Conclusions
1) Normal S-S bond formation can be expected. 2) Target proteins can be purified directly from the media without need for bacterial lysis. 3) A large amount of target protein can be obtained because they are secreted into the media rather than sequestered in the bacterial cytoplasm. 4) Some proteins that readily form inclusion bodies when expressed in E. coli are more likely to maintain their normal folding structure during dilution into the culture media. 5) Even cytotoxic proteins that exhibit toxicity inside the cells can potentially be produced efficiently using the Brevibacillus Expression System by being secreted into the extracellular medium. 6) The Brevibacillus Expression System offers an affordable alternative to eukaryotic systems. Although eukaryotic expression systems such as insect cells are primarily used for the secretory production of recombinant protein at present, eukaryotic systems requires both sophisticated protocols and expensive equipment compared with the E. coli expression system. From that perspective, the Brevibacillus Expression System could be recommended as an alternative method for producing proteins that cannot be easily expressed by E. coli. The Brevibacillus system is compatible with culture equipment and materials used for E. coli expression. Additionally, NMR structural analysis of secreted proteins produced with the Brevibacillus Expression System may become more attractive in the future.
1. Udaka, S., Yamagata, H. (1993) High-level secretion of heterologous proteins by Bacillus brevis. Methods Enzymol. 217:23-33. 2. Miyauchi, A., Ozawa, M., Mizukami, M., Yashiro, K., Ebisu, S., Tojo, T., Fujii, T., Takagi, H. (1999) Structural conversion from non-native to native form of recombinant human epidermal growth factor by Brevibacillus choshinensis. Biosci. Biotechnol .Biochem. 63(11):1965-1969. 3. Yashiro, K., Lowenthal, J. W., O’Neil, T. E., Ebisu, S., Takagi, H., Moore, R. J. (2001) High-level production of recombinant chicken interferongamma by Brevibacillus choshinensis. Protein Expr. Purif. 23(1):113-120. 4. Tanaka, R., Mizukami, M., Ishibashi, M., Tokunaga, H., Tokunaga, M. (2003) Cloning and expression of the ccdA-associated thiol-disulfide Oxidoreductase (catA) gene from Brevibacillus choshinensis: stimulation of human epidermal growth factor production. J. Biotechnol. 103(1):1-10. 5. Tanio, M., Tanaka, T., Kohno, T. (2008) 15N isotope labeling of a protein secreted by Brevibacillus choshinensis for NMR study. Anal. Biochem. 373(1):164-166. 6. Tanio, M., Kohno, T. (2009) Histidine regulated activity of M-ficolin. Biochem. J. 417(2):485-491. 7. Tanio, M., Tanaka, R., Tanaka, T., Kohno, T. (2009) Amino acid-selective isotope labeling of proteins for nuclear magnetic resonance study: Proteins secreted by Brevibacillus choshinensis. Anal. Biochem. 386(2):156-160. 8. Tanio, M., Kondo, S., Sugio, S., Kohno, T. (2006) Overexpression, purification and preliminary crystallographic analysis of human M-ficolin fibrinogen-like domain. Acta Crystallogr. Sect. F Struct. Biol. Cryst. Commun. 62(Pt. 7):652-655. 9. Tanio, M., Kondo, S., Sugio, S., Kohno, T. (2007) Trivalent recognition unit of innate immunity system: crystal structure of trimeric human M-ficolin fibrinogen-like domain. J. Biol. Chem. 282(6):3889-3895. 10 Tanio, M., Kondo, S., Sugio, S., Kohno, T. (2008) Trimeric structure and conformational equilibrium of M-ficolin fibrinogen-like domain. J. Synchrotron Radiat. 15(Pt. 3):243245.
However, in our experience, some proteins were expressed at low levels and others could not even be cloned into the vector. Moreover, in order to improve the yield of the obtained protein, increasing TALON resin volume and re-applying the flowthrough is also required. We believe this implies that some 2SYNm and TMNm medium components may prevent absorption of His-tags. In the future, we expect that additional studies on both advantages and drawbacks of Brevibacillus Expression System will lead to further developments and improvements of this expression system.
Figure 3. 15N stable isotope labeling with Brevibacillus Expression System. 1H-15N HSQC NMR spectrum results of A) FD1 protein
with 15N-labeled amino acids, B) FD1 protein with [α-15N] labeled Ala, and C) FD1 protein with [α-15N] labeled His. It has been known that FD1 protein has 2 S-S bonds (Fig.2). This experiment was perfomed with a FD1 variant. (F127S/L128S) stays as monomer in solutions, because wild-type FD1 forms trimers.
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19
6 Application Notes
The production of secreted recombinant protein using the Brevibacillus Expression System has several advantages:
References
The pCold TF Protein Expression System Produces Soluble, Active Protein in E. coli
Application Notes
6
The elucidation of protein structure and function continues to be important in the post-genomic era. An efficient protein production system is critical for obtaining large amounts of correctly folded recombinant protein for study. E. coli expression systems are used extensively for production of recombinant proteins, and have two major advantages over other expression systems: (1) ease of use, and (2) low cost. However, some recombinant proteins do not fold correctly during expression in E. coli, resulting in deposits of inactive insoluble protein termed “inclusion bodies”.
Materials and Methods
Series of pCold Vectors In collaboration with Prof. Masayori Inouye (University of Medicine and Dentistry of New Jersey), Takara Bio has developed the pCold DNA Vectors, a series of novel protein expression vectors. The pCold Vectors provide increased in vivo protein yield, purity, and solubility of expressed recombinant proteins using “cold shock” technology. The cspA (cold shock protein A) promoter and related elements have been incorporated into these vectors to up-regulate target protein production at lowered incubation temperatures (37°C-15°C). This temperature drop also suppresses expression of other cellular proteins, represses protease activity, and temporarily halts overall cell growth. This process allows expression of target proteins at high yield, high purity (up to 60% of cellular protein), and increased solubility as compared with conventional E. coli expression systems. Co-expression of one or more chaperone proteins during expression of a heterologous target protein has proven effective for obtaining higher amounts of soluble recombinant protein (see Takara’s Chaperone Plasmid Set (Cat. # 3340)). This procedure, though, lacks the convenience of a single transformation step.
pCold TF Vectors Takara’s pCold TF DNA Vector is a fusion cold shock expression vector that expresses a molecular chaperone (Trigger Factor (TF)) as a soluble tag. Trigger Factor is a prokaryotic ribosome-associated chaperone protein (48 kDa) that facilitates co-translational folding of newly expressed polypeptides. Because of its E. coli origin, TF is highly expressed in E. coli expression systems. The pCold TF DNA Vector consists of the cspA (cold shock) promoter plus additional downstream sequences including a 5’ untranslated region (5’ UTR), a translation enhancing element (TEE), a His-Tag sequence, and a multiple cloning site (MCS). A lac operator is inserted downstream of the cspA promoter to ensure strict regulation of expression. Additionally, recognition sites for HRV 3C Protease, Thrombin, and Factor Xa are located between the TF-Tag and the MCS to facilitate tag removal from the expressed fusion protein. Most E. coli strains can serve as expression hosts.
20
pCold TF DNA Vector combines high-yield cold shock expression technology with Trigger Factor (chaperone) expression in a single vector to facilitate correct protein folding, thus enabling efficient soluble protein production for otherwise intractable target proteins. The following experiment compares results generated using 1) pCold I, 2) pCold I co-expressed with a Chaperone plasmid, 3) pCold TF, or 4) T7 promoter constructs to express various proteins.
pCold DNA I and pCold TF DNA cloning and expression procedures* were conducted as described in this experimental overview: 1) Insert the target gene to the multiple cloning site of the pCold DNA vector for expression. 2) Transform the E. coli host strain (e.g. BL21) with the expression plasmid and select for ampr transformants. 3) Inoculate the transformants into medium including 50 µg/mL of ampicillin, and incubate with shaking at 37°C. 4) At OD600= 0.4 - 0.5, refrigerate the culture at 15°C (without shaking) for 30 minutes. 5) Add IPTG to a final concentration of 0.1-1.0 mM, then continue incubation with shaking at 15°C for 24 hours. 6) Collect the cells and confirm the expression of the target protein by SDS-PAGE of soluble and insoluble fractions or by activity assay. Expression from T7 promoter-driven vectors was performed using a standard protocol utilizing IPTG induction and subsequent culturing at 37°C. *Cultivation/induction conditions (culture medium, aeration, timing of induction, concentration of inducer, cultivation time after induction) should be optimized for each target protein.
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Results and Discussion kDa
pCold TF 1 2
pCold + Chaperone 1 2
pCold 1 2
97 66
1. Cell extract solution 2. Soluble fraction target protein co-expressed trigger factor
*
31
Figure 1: Expression of Soluble Protein A Using the pCold TF Expression System
Trx
GST
Nus
1 2 3 1 2 3 1 2 3
pCold pCold I pCold + Chaperone TF 1 2 3 1 2 3 1 2 3
kDa
45
Figure 1 shows the successful production of enzyme protein A using the pCold TF system. Upon expression, this protein (estimated molecular weight 29 kDa) was not observed as a discernable band with either the T7 expression system or even with pCold I (by either individual expression or chaperone co-expression). However, the expression of the target protein and target plus tag (29 kDa and 52 kDa) was observed using pCold TF, and most of the obtained protein was in soluble form. Subsequent assays confirmed that the expressed enzyme A retains activity even as a fusion protein (data not shown).
22
97 66
Protein expression using the pCold TF Expression Vector was compared with protein expression using (1) the pCold DNA I Vector alone, (2) co-expression using the pCold DNA I Vector with Takara Bio’s Chaperone Plasmid pTf16, and (3) a T7 promoter expression system which included other solubilization-promoting tags.
1. Cell extract solution 2. Soluble fraction 3. Insoluble fraction
31 22
Figure 2 shows improved expression of soluble protein B using pCold TF. Expression of soluble enzyme protein B (M.W: ~63 kDa) was not observed using either pCold DNA I alone or pCold I co-expressed with chaperone proteins, nor with a T7 expression vector that included other tags for solubilization (Trx Tag [~12 kDa], Nus Tag [~55 kDa], and GST Tag [~26 kDa]). However, when the pCold TF DNA Vector was used, the target protein was observed at an expression level much higher than that achieved with other systems and tags, and most of the expressed target protein was observed in the soluble fraction. (Note: due to the presence of various tags, target protein molecular weight appears larger and more variable than its actual size). In summary, the pCold TF expression system offers several advantages including convenience, high yield, and high purity. The pCold TF system is suitable for efficient soluble protein expression in E. coli of otherwise intractable target proteins.
Figure 2: Increased Expression of Soluble Protein B Using the pCold Expression System
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21
6 Applications Notes
*
45
T7 1 2
Unfolding the Potential of Proteins New refolding technologies are essential for tomorrow’s recombinant proteins By Joby Marie Chesnick
In vivo, small polypeptides often fold spontaneously into their correct configuration. However, longer polypeptides have a greater likelihood of folding more slowly and have greater potential to form partially folded intermediate structures and aggregates that are non-functional. The timing of polypeptide folding can also influence the success rate of correct protein folding. For example, outcomes may very if protein folding is concomitant with ribosomal synthesis or if it is delayed until transport into the cytoplasm or to cellular organelles. One of the largest problems encountered by scientists attempting to express recombinant proteins in bacteria is the formation of inclusion bodies, insoluble aggregates of misfolded polypeptides that are produced as the bacterium quickly synthesizes large quantities of the foreign protein. These misfolded protein aggregates must be unfolded and refolded to assume their correct 3-dimensional structure before further study or production can proceed. As research efforts have continued to shift from the investigation of gene structure to the study of protein structure and function, the importance of studying recombinant proteins has increased. Robust methods for producing accurately folded structures are therefore essential.
Molecular chaperones More than 40 years ago, Ferruccio M. Ritossa from the International Laboratory of Genetics and Biophysics in Naples, Italy, first discovered the heat shock response of certain proteins through observation of a new puffing pattern in Drosophila buschii salivary gland polytene chromosomes (1). This puffing was indicative of increased gene expression of these proteins, which were later termed heat shock proteins (HSPs). HSPs and related proteins (e.g. DnaK, Hsp40, GrpE; GroEL/S; trigger factor; prefoldin CCT; SecB; ClpA) are called “molecular chaperones” because they help ensure that polypeptides assume the correct conformation. One of the most important functions of chaperones is in aiding the protein-folding process. Because molecular chaperones coevolved with polypeptides—the presence of highly conserved sequence data for chaperones and representation of these molecules in every major taxon, including eukaryotes, eubacteria, archaea, and viruses, suggests that chaperones are ancient molecules—they provide for a controlled cellular mechanism by which proteins can reach their most thermodynamically stable 3-dimensional conformation. Chaperones accomplish controlled protein folding either by directly binding to conserved domains in a nascent polypeptide and preventing interactions with other adjacent protein domains or by providing, through their own 3-dimensional structure, a space that allows controlled polypeptide folding (2). Folding typically occurs via several binding and release events with the polypeptide, which are mediated by the hydrolysis of ATP. But even with all of the chaperone machinery present in a cell, 30% or more of all synthesized polypeptides cannot fold correctly to form functional proteins (3). Nonetheless, when there is need to refold proteins to their correct form, molecular chaperones are essential tools.
22
100
Relative activity, %
Application Notes
6
The development of novel enzymes and proteins relies upon an understanding of their intrinsic structures. A protein’s 3-dimensional structure dictates the positions of exposed reactive groups as well as hidden hydrophobic residues, thereby defining its biological activity. Three-dimensional structure can also influence processes such as protein trafficking to and between cellular organelles. It is well-documented that several human diseases including Alzheimer’s Disease, Parkinson’s Disease, and Cystic Fibrosis arise due to misfolded cellular proteins.
Tween 60 Tween 40
80 60 40 20 0
0
0.2
0.4
0.6
0.8
1.0
Final concentration of CA, %
Figure 1. Comparison of citrate synthase activity following protein refolding using cycloamylose and Tween 40 or Tween 60. Full (100%) activity is achieved for refolding of citrate synthase using as little as 0.6% cycloamylose with Tween 60.
Refolding strategies The most common strategy currently used to recover active recombinant protein in vitro from isolated inclusion bodies consists of a three-step process: (1) isolation and washing of the inclusion bodies, (2) solubilization (i.e., denaturation and unfolding) of the protein aggregates, and (3) correct refolding of the solubilized protein. The first two of these steps typically can be performed with high efficiency. However, misfolding and aggregation of the solubilized protein may complicate the last step. A number of commercial kits are available for improving the refolding conditions of inclusion bodies, including Fold-It, developed by Hampton Research; Pro-Matrix Protein Refolding Kit from Pierce; and Novagen’s Protein Refolding Kit. Their components include a denaturant to solubilize the inclusion bodies, one or more small-molecule detergents to maintain the protein in an unfolded configuration and allow refolding through transient interactions with the protein, and various buffers that differ in parameters that influence the refolding process, such as pH, redox concentration, and ionic strength. The degree of successful refolding obtained by the “dilution additive” strategy of these kits depends on the buffer properties, as well as protein concentration and temperature. The Refolding CA Kit from Takara Bio offers a novel approach to the dilution additive method. By using an artificial chaperone—highly polymerized cycloamylose (CA)— the kit provides correct refolding of aggregated proteins with high effiency. CA aids refolding through its structure, a single helical V-amylose conformation containing an anhydrophilic channel-like cavity (4). Inclusion complexes with other molecules such as detergents can be formed in this space, preventing protein aggregation. In contrast to the conventional dilution additive method, CA supports stable interactions between the protein and detergent, and then strips the detergent from the protein–detergent complex to initiate refolding (5). This scenario results in greater refolding efficiency. CA can be used in combination with many different types of detergents and is compatible with redox reagents. In addition, CA can interact with peptides of multiple sizes, is highly soluble and has a long shelf life in aqueous solution, and accomplishes protein
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refolding in a short time (i.e., a few hours to overnight). Figure 1 shows the amount of citrate synthase activity recovered following enzyme refolding using CA. In this application, full enzyme activity was obtained using 0.6% CA with Tween 60 detergent (polyoxyethylene sorbitan monostearate) or 1% CA with Tween 40 (polyoxyethylene sorbitan monopalmitate).
6
The Chaperone Plasmid Set, developed by HSP Research Institute and introduced into the market by Takara Bio, contains five chaperone-team-containing plasmids for increasing yields of soluble foreign proteins in vivo. These plasmids carry a pACYC origin of replication plus a chloramphenicol resistance gene, which allows their use with ColE1type plasmids containing an ampicillin resistance gene. Clones may be selected based on presence of both a chaperone plasmid and a plasmid containing a target gene of interest. Figure 2 shows increased amounts of soluble target protein (bacterial protein D), with a concomitant decrease in insoluble protein, obtained by co-expression of chaperones from Takara’s pG-KJE8 chaperone plasmid vector. For obtaining high yields of recombinant proteins, Takara Bio offers plasmids that utilize the cold-shock protein A (cspA) gene promoter system for gene expression. This system induces protein expression at low temperatures (15 °C), which suppresses the synthesis of most other proteins and lowers potentially destructive cellular protease activity. As a result, up to 60% of all expressed cellular protein is the desired target. Additionally, high-efficiency metabolic labeling of the protein is possible to facilitate structural analysis. Large-scale culture and affinity purification (up to 400 mg of protein per liter of culture) is achievable, making cold shock protein expression suitable for commercial applications.
Figure 2. Soluble protein production with co-expression of chaperone genes from Takara’s chaperone plasmid vectors with a bacterial gene. A bacterial gene was present at relatively low level in the soluble fraction in absence of co-expressed chaperones, but solubility was enhanced when chaperones were co-expressed. Arrow indicates target protein.
The pCold Vector series from Takara Bio allow insertion of a foreign target gene into a vector for expression using the cspA promoter. The combination of chaperone plasmid and cold-shock vector technologies provides a system by which high specificity, highyield production of soluble expressed foreign proteins is routinely possible at affordable costs. The availability of new and modified proteins and enzymes for medical and biological research will depend to a large extent on the development of quick, reliable, and costeffective refolding methods. Reprinted with permission from Modern Drug Discovery, July 2004, 7(7), 67. Copyright 2004 American Chemical Society.
References: (1) Ritossa, F. (1962) A new puffing pattern induced by temperature shock and DNP in Drosophila. Experientia 18:571-573. (2) Hartl, F.U.; Hayer-Hartl, M. (2002) Molecular chaperones in the cytosol: from nascent chain to folded protein. Science 295(5561):1852-1858. (3) Schubert, U., et al. (2000) Rapid degradation of a large fraction of newly synthesized proteins by proteasomes. Nature 40(6779):770-774. (4) Machida, S., et al. (2000) Cycloamylose as an efficient artificial chaperone for protein refolding. FEBS Letters 486(2):131-135. (5) Daugherty, D.L., et al. (1998) Artificial chaperone-assisted refolding of citrate synthase. J.Biol. Chem. 273(51):33961-33971.
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23
Application Notes
An alternative in vivo method for obtaining increased soluble expressed protein relies on chaperone-containing plasmid vectors. In this case, a “team” of chaperone genes, such as DnaK-DnaJ-GrpE or others, is engineered into a plasmid vector and placed under control of either an araB or Pzt-1 promoter, with induction by L-arabinose or tetracycline, respectively. Chaperones are then co-expressed with the target protein of interest in E. coli. Separate expression of chaperones and target proteins can be accomplished if the target gene is placed under the control of a different promoter (e.g., lac).
SPP System™ (Single Protein Production System) 1) What strategies can be used to facilitate production of soluble protein of interest when it is initially expressed in insoluble form? Possible strategies are:
FAQs
7
• C hange the time at which IPTG is added to induce the expression. It may be necessary to optimize the most ideal timepoint, which can range from early to late logarithmic phases of the culture. • Reduce the concentration of IPTG (down to 0.1 mM). • Change the strain of host E. coli. • Extract the cultured cells by sonication in a buffer containing 0.1 to 1% of detergent (for example, octylglycoside, NP-40, Triton X-100, etc.)
2) How do I select the type of Cold-shock expression vector for SPP to use? The Translation Enhancing Element (TEE) facilitates translation when using pCold I (SP-4), pCold II (SP-4) and pCold III (SP-4). Proteins expressed using pCold I (SP-4) and pCold II (SP-4) include His-tags and can be affinity-purified with Ni columns. If you do not desire the presence of any additional amino acid sequences at the N-terminus of the protein of interest, the pCold I (SP-4) vector is recommended, as it allows cleavage of the Tag sequence with Factor Xa. Alternatively, pCold IV (SP-4) lacks both TEE and Tag sequences.
3) What quantity of the protein of interest will be produced from 1 L of culture? The expression level usually ranges from several mg to several tens of mg per L, although it varies for each protein of interest. A 3-L culture can typical result in miligrams of purified protein, provided the protein of interest can be detected by SDS-PAGE followed by Coomassie brilliant blue (CBB) staining. In the MazF co-expression system, expression of new endogenous host proteins is suppressed except for target protein. This results in highly specific production of target protein with target protein comprising up to 90% of total protein, but may result in growth stress for E.coli itself. Accordingly the expression yield of a target protein may decrease compared to general expression with pCold DNAs.
24
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pCold Expression Vectors 1) What are suitable competent cells for pCold vectors for protein induction and plasmid storage? Takara’s pCold Vectors utilize an E. coli cold-shock gene promoter. Therefore, most E.coli strains can be used as a host for protein induction with these vectors. The following E. coli strains have been tested and are suitable for use with the pCold Vectors:
2) Where can I obtain sequence information for the pCold Vectors? Sequencing information for all pCold Vectors has been deposited in GenBank and is available from the NCBI website (http://www.ncbi.nlm.nih.gov/). GenBank Accession Numbers for each of the pCold Vectors are listed below:
pCold Vector pCold I pCold II pCold III pCold IV pCold TF
GenBank Accession No. AB186388 AB186389 AB186390 AB186391 AB213654
3) Can you tell me exactly where the cleavage site for Factor Xa is located? Is it at Arg-His? Factor Xa cleaves at the C-terminal side of last amino acid (Arg) of the recognition sequence Ile-Glu-Gly-Arg. Thus, the Arg-His will be cleaved.
4) How much Factor Xa would be required to cleave 1 mg of expressed protein? Takara Bio scientists have used Factor Xa, Restriction Grade (Novagen) for cleavage. Cleavage conditions vary from protein to protein. For example, Takara Bio scientists have performed cleavage using 1 U of Factor Xa for 3 µg of a 60 kDa protein during an overnight incubation at 4°C. Be sure to optimize the cleavage conditions for each target protein on a small scale prior to scaling up reactions.
7
To use the translation enhancing element (TEE) provided in pCold III, the target gene sequence must be in frame with the TEE sequence. The ATG start codon in the TEE is used for protein expression. Thus, using this vector, your expressed protein will be a fusion protein. To express only the target protein without TEE, use pCold IV instead.
FAQs
• BL21 strains • Rosetta (good for eukaryotic genes) • Origami (good for eukaryotic genes, but grow very slowly) • JM109 JM109 or DH5α cells are typically used to construct expression plasmids which are inserted with a target gene. Note, however, that we recommend storing the pCold Vectors as isolated plasmid DNA, rather than in transformed cells.
5) We intend to use pCold III for expression of our protein. Our target gene sequence must contain an ATG start codon so that it can also be used with our PCR primers. However, since the TEE element provided on pCold III also has an ATG start codon, will this situation (i.e. the presence of an ATG start site in both TEE and our target gene sequences) pose a problem for expression?
We recommend using the Nde I restriction site in the MCS for fusion of an insertion (gene) sequence with a start codon. For example; If the sequence of the target gene is this:
ATG AGC GAT AAA ATT ATT CAC.....
Met-Ser-Asp-Lys-Ile-Ile-His-....
and if pCold III containing TEE is used, then fuse the gene into the Nde I site (CATATG) as shown below, where the gene ATG start site is underlined: Nde I site ...ATG AAT CAC AAA GTG CAT ATG AGC GAT AAA ATT ATT CAC.....
The expressed protein will then be: Met-Asn-His-Lys-Val-His-Met-Ser-Asp-Lys-Ile-IleHis-...
For PCR amplification of the target gene, design the PCR forward primer considering the points presented above.
6) I recently purchased the pCold I DNA vector and I was wondering what antibody if any could be used with the recombinant proteins produced using this vector? Since most proteins produced using the pCold Vectors will include a His-tag, Takara Bio recommends using a His-tag antibody. Clontech offers three His-tag antibodies: 6xHis Monoclonal Antibody (Albumin-Free) (Cat. #631212), 6xHN Polyclonal Antibody (Cat. #631213), and 6xHis mAb-HRP Conjugate (Cat. #631210). See the Clontech Related Products section on page 31 for ordering information.
7) Have any of the pCOLD vectors been used for expression in a cell-free system? Takara does not have information about use of pCold Vectors for expression in a cell-free system. However, it is quite possible that general cell-free systems are not suitable for use with the pCold Vectors since these systems are usually optimized for expression at 37°C.
Clontech, A Takara Bio Inc. Company • www.clontech.com/takara
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Refolding CA Kit
FAQs
7
1) What is the difference between the two different sizes of Refolding CA Kits?
3) Can disulfide bond-reforming components, such as reduced/ oxidized glutathione, be used with Takara’s Refolding CA Kit?
The smaller Refolding CA Kit (Cat. # 7350) has enough reagents for 25 refolding reactions. This product is sold as an initial kit for refolding smaller amounts of proteins (typically not more than 0.24 mg protein per reaction; each reaction uses ~24 µl of a 10 mg/ml inclusion body solution) and for testing refolding conditions using the provided detergents with the CA. The small kit includes denaturant (8 M GdmCl), 4 M DTT, 4 different surfactants (1% Tween 40, 1% Tween 60, 1% CTAB, 1% SB3-14), 100 mM DL-Cystine, and 3% cycloamylose (CA). Note that urea can also be used with our kit, although it is not included as a component. Please refer to the product manual for details regarding the kit protocol.
Yes, disulfide bond-forming reagents can be used with the Refolding CA Kit. Note that our Kit contains DL-Cystine for disulfide bond reformation. The following reference for use of reduced/ oxidized gluthathione may be helpful:
The large kit (Cat. # 7351) is intended for use after optimal refolding conditions have been determined using the small kit, and/or when there is need to refold larger amounts of protein. The large kit includes 10-fold more denaturant (8M GdmCl)(=20 mL) and cycloamylose (CA)(= 120 mL) than the small kit. The large kit does not include the detergents, DTT, or DL-Cystine. These reagents are available separately from chemical reagent suppliers (e.g., Sigma Aldrich).
2) Could you provide the name of a source for DTT, DL-cysteine and SB3-14 detergent to be used with the Refolding CA Kit? I want to make sure that we purchase exactly the same items that came in the small Refolding CA Kit. Takara uses the following products for this kit:
Component
Supplier
Catalog #
DTT
nakalai
14128-91
Sigma
D5545
DL-Cystine
nakalai
10316-41
Sigma
C8630
SB3-14
Sigma
T0807
(Note: SB3-14 is registered as N-Tetradecyl-N,N-Dimethyl-3-Ammonio-1Propanesulfonate at Sigma) Products from the Japanese manufacturer “nakalai” may not be available in your geographic location. However, Sigma’s products (listed above in parentheses) are comparable for use with this kit.
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Machida, S., et al. (2000) Cycloamylose as an efficient artificial chaperone for protein refolding. FEBS Lett. 486(2):131-135. This reference recommends using GSH:GSSH at a ratio of 5:1.
4) Are all of the detergents removed from solution after addition of cycloamylose (CA)? The surfactants form a precipitant with CA, and the majority of these detergents are thus removed after the final centrifugation step. However, a small amount of residual surfactants remain in the final refolding protein solution. To remove free surfactants, we suggest using BioBeads (Bio-Rad). Add a 1/5 volume of BioBeads to the final refolding protein solution and stir the mixture for 2 hours. After stirring, the Bio Beads are removed and the solution is recovered. Using this method, more than 95% of ionic surfactants and more than 90% nonionic surfactants are removed. It is, however, difficult to completely remove ALL traces of surfactants in the refolding protein solution.
5) I do not get a precipitate in the very last step of the protocol when I use either Tween 40 or Tween 60 with the CA, but I do get a precipitate if I use either of the other two detergents. Why am I not getting a precipitate with the Tween 40 or Tween 60? On rare occasions Tween 40 or Tween 60 will sometimes fail to form a precipitate during the last step; however, good refolding results are still obtained even though a white precipitate is not observed. The exact buffer or salt composition and concentration may influence precipitate formation. However, at present there is not a clear explanation as to why visible precipitate formation may be variable. If you do not observe a precipitate, continue with the procedure regardless. You should perform centrifugation at 15,000 rpm for 10 minutes, and then collect the supernatant to assess the protein activity.
Clontech, A Takara Bio Inc. Company • www.clontech.com/takara
Chaperone Plasmid Set 1) We are trying to express a protein which contains a few rare codons. This means that we need to express the protein in special strains as Stratagene BL21 Codon plus or Novagen Rosetta. Is there any incompatibility of your Chaperone Plasmids with these kinds of strains? Novagen Rosetta and Stratagene BL21 Codon plus are the only E. coli strains that encode rare codons. Unfortunately, these strains are chloramphenicol-resistant. Because Takara’s Chaperone plasmids all contain a pACYC ori and chloramphenicol-resistance gene, these strains cannot be used as a host for co-expression with the chaperone plasmids.
Takara has successfully folded a 70 kDa protein using a combination of pCold Vectors (which utilize cold shock expression technology for high yield-high purity protein expression) in conjunction with one of our Chaperone Plasmids.
3) After I have used the Chaperone Plasmids for correct folding of my target protein in vivo, can I successfully purify the expressed target protein without co-purification of the chaperone protein? Normally I use a His-tag to purify my expressed proteins, but a Histag can interact with dnaK. Takara often uses His-tags to purify target proteins folded in vivo using the Chaperone Plasmids, and has never experienced chaperone protein contamination, such as with dnaK, with target proteins that are purified using His-tag/His-bind resin. For target protein purification using His-bind resin, be sure to use an appropriate binding buffer for purification with the proper concentration of NaCl and imidazole. However, since chaperone proteins such as DnaK or DnaJ do tend to non-specifically bind to the resin, it may be somewhat difficult to purify GST-fusion proteins by affinity column purification. In this case, we recommend using buffer that contains ATP and Mg2+, or using an ATP-agarose resin for release of protein from the column. For further details of these procedures, please refer to the following references: MYu, S., et al. (1992) Involvement of the chaperonin dnaK in the rapid degradation of a mutant protein in Escherichia coli. EMBO J. 11(1):71-77. Zylicz, M., et al. (1984) Purification and properties of the Escherichia coli dnaK replication protein. J. Biol. Chem. 259(14):8820-8825.
Takara uses mainly BL21 or BL21 (DE3) as host strains with these plasmids. Although Takara does not have specific recommendations for E.coli strains, some generalizations may be made regarding the optimal combination of target expression vector and chaperone team used. For example, the chaperone teams of groES-groEL and/ or dnaK-dnaJ-grpE may be more useful than tig (Trigger Factor) to fold proteins which are expressed using pET vectors, regardless of the kind of target protein. However, in general, we recommending testing all five chaperone plasmids for each target protein in order to optimize results.
6) Is DNA sequence information available for the Chaperone Plasmids? Takara’s Chaperone Plasmids are currently available under a license agreement between TAKARA BIO INC. and HSP Research Institute, Inc. As a result, to obtain the sequences of any of these plasmids, customers are required to complete and submit a Sequence Request Form. This form is available on the Chaperone Plasmid Set product page at www.clontech.com/takara. Click on the orange box in the ordering area at the bottom of the Chaperone Plasmid Set product page to obtain the form. Upon approval of the request, the sequence information will be forwarded to you.
7) Are all chaperone genes encoded on an individual Chaperone Plasmid expressed in the same ratio? Chaperone genes which are under the control of the same promoter in an individual Chaperone Plasmid will be expressed in the same ratio. For example, DnaK, DnaJ and GrpE, which are encoded on the plasmid pKJE7, will all be expressed in the same ratio in cells. The following references provide SDS-PAGE data for chaperone co-expresssion: Nishihara, K., et al. (1998) Chaperone coexpression plasmids: differential and synergistic roles of DnaK-DnaJ-GrpE and GroEL-GroES in assisting folding of an allergen of Japanese cedar pollen, Cryj2, in Escherichia coli. Appl. Environ. Microbiol. 64(5):1694-1699. Nishihara, K., et al. (2000) Overexpression of trigger factor prevents aggregation of recombinant proteins in Escherichia coli. Appl. Environ. Microbiol. 66(3):884-889.
8) Is it possible to use the Chaperones Plasmids to aid in vitro translation? Takara’s Chaperone Plasmid Set is designed to aid only in the folding of expressed proteins in vivo. We do not have any information as to whether the Chaperone Plasmid Set can be used to aid in vitro translation.
4) Are restriction maps available for any of the vectors in the chaperone plasmid set? Restriction maps for Takara’s Chaperone Plasmids are not available. However, information is available for those restriction enzymes which cut each plasmid only once. These enzymes are listed below. The number in ( ) is the 5’ terminal of each digestion site. • -pKJE7; Kpn I (5660), Nhe I (6606), Sac I (4157), Sca I (410), Spe I (5632) • -pG-KJE8; Nhe I (10608), Sac I (4157), Sca I (410), Xho I (8181) • -pGro7; Bgl II (3929), EcoR I (5462), Hind III (3941), Nhe I (4880), Sca I (410), Sma I(3832), Xba I (4040) • -pG-Tf2; BamH I (1979), Hind III (8208), Nhe I (7268), Sal I (4493), Sca I (6271), Sma I (2786), Spe I (4511), Xho I (1) • -pTf16; BamH I (3323), Bgl II (1535), Hind III (1523), Kpn I (1529), Nhe I (583), Sca I (4601), Sma I (2534), Xba I (1424) Clontech, A Takara Bio Inc. Company • www.clontech.com/takara
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7 FAQs
2) We have a 64 kDa protein that we have expressed in E. coli. After induction there is a reasonable amount of soluble protein, but most of this protein is not functional, probably because is not properly folded. Can Takara’s Chaperone Plasmids help us to overcome problems associated with expressing a larger protein such as this one?
5) Has Takara tested any strains of E. coli with these plasmids? Do they have a recommendation for strains that work best with the plasmids?
Takara’s Protein Sequencing and Analysis Products Takara offers a wide variety of Protein Fragmentation products as well as N-terminal deblocking and sequence determination and C-terminal sequence determination products. The fragmentation products are used for analysis of the primary structure of proteins and peptides.
Product Name
N-terminal and C-terminal analysis
Pfu Aminopeptidase I
Pfu Pyroglutamate Aminopeptidase
Pfu Methionine Aminopeptidase
Fragmentation of Proteins
Pfu N-acetyl Deblocking Aminopeptidase (Ac-DAP)
Protease Inhibitor
Protein Sequencing and Analysis Products
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Application • Liberates N-terminal amino acids up to X-Pro from proteins and peptides • Removal of pyroglutamic acids from the N-terminal of proteins and peptides • Deblocking of N-terminal pyroglutamates of proteins and peptides for sequence analysis using Edman degradation
• Liberates the N-terminal methionine residues from proteins and peptides
• N-Terminal deblocking • N-terminal sequence analysis of blocked proteins or peptides
Description Pfu Aminopeptidase I is a thermostable exo-type aminopeptidase, isolated from Pyrococcus furiosus and produced as a recombinant protein, which liberates the N-terminal amino acid from proteins and peptides. Pfu Pyroglutamate Aminopeptidase liberates the N-terminal pyroglutamic acid from proteins and peptides. This enzyme may work well with some intact, non-denatured proteins and the denaturation step may be unnecessary in these instances. Pfu Methionine Aminopeptidase specifically liberates only the N-terminal methionine residue from Met-X-Y when X is Ala, Gly, Ser, Thr, Pro or Val. This enzyme does not liberate the N-terminal Met when the N-terminus sequence is Met-Met-Y or Met-Met-Met-Y. It is not active toward formyl-methionine. Pfu N-acetyl Deblocking Aminopeptidase (Ac-DAP) is a unique exotype aminopeptidase that first liberates blocking groups, such as formyl, acetyl, and myristyl, and then releases the first and subsequent amino acids from proteins and peptides until it reaches the first X-Pro bond.
Arginylendopeptidase
• Fragmentation of proteins and peptides required from primary structure analysis
Arginylendopeptidase cleaves peptide bonds at the carboxyl side of arginine residues found in proteins and peptides. Arginylendopeptidase is also known as mouse submaxillary protease D or as mouse EGF binding protein C.
Asparaginylendopeptidase
• Fragmentation of proteins and peptides required for primary structure analysis
Asparaginylendopeptidase specifically cleaves peptide bonds on the carboxyl side of asparagine residues found in proteins and peptides. Glycosylated asparagine residues are not cleaved.
Endoproteinase Asp-N
• Fragmentation of proteins and peptides required for primary structure analysis
Endoproteinase Asp-N is a metalloprotease that hydrolyzes peptide bonds on the amino side of Asp and Cys oxidized to cysteic acid. If cysteine is reduced or alkylated, the enzyme will cleave only the amino side of Asp residues.
Pfu Protease S
• Fragmentation of proteins and peptides required for primary structure analysis
Pfu Protease S is an endo-type serine protease with broad recognition of native and denatured proteins. Cleavage occurs mainly on the carboxy side of peptide bonds of hydrophobic amino acid residues.
• Calpain protease inhibitor
Calpastatin is an endogenous protease inhibitor that acts specifically on calpain calcium-dependent cysteine protease. It consists of four repetitive sequences of 120 to 140 amino acid residues (domains I, II, and IV), and an N-terminal non-homologous sequence (L). The product consists of highly purified recombinant human calpastatin domain I .
Calpastatin
Clontech, A Takara Bio Inc. Company • www.clontech.com/takara
PrimeSTAR® GXL DNA Polymerase PrimeSTAR® GXL DNA Polymerase PrimeSTAR® GXL DNA Polymerase
R050A R050B
250 Units 1,000 Units
• Successful, Robust, High-Yield PCR regardless of Conditions • Long PCR: Amplify products up to 30 kb (human genomic DNA), 40 kb (lambda DNA), or 13.5 kb (human cDNA) • Use on Standard and Challenging Templates alike: Outstanding performance on GC-rich or AT-rich templates and targets containing repeats • Can be used with Samples Containing Excess Nucleic Acid: Tolerates a wide range of template quantity, including high levels of template that inhibit other high-fidelity DNA polymerases • Next Generation Sequencing (NGS) studies involving deep sequencing of the same region in many samples
PrimeSTAR® GXL Company I Company T M 1 2 3 4 M M 1 2 3 4 M 1 2 3 4 M
Features • Highest Processivity: among commercially available high-fidelity DNA polymerases • Antibody-Mediated Hot-Start Formulation • Proven Performance: as reported in peer-reviewed literature
Comparison of Amplification of GC-rich targets using PrimeSTAR GXL and other commercially available high-fidelity DNA polymerases. Excellent results were achieved using PrimeSTAR® GXL DNA Polymerase without requiring special buffers or reaction conditions.
Description Capable of outstanding performance for both routine high-fidelity PCR and challenging templates or reaction conditions, PrimeSTAR GXL DNA Polymerase is the most robust high-fidelity PCR enzyme commercially available. It provides high yield, high specificity, and high accuracy for not only standard reactions, but also excels in PCR with GC-rich templates, in the presence of eXcess template, and for amplification of Long products up
Template: Human genomic DNA (100 ng / 50 μl Template: T. thermophilus HB8 genomic DNA reaction) 1. APOE gene 746 bp (GC content 74%) 2. TGF (10 ng / 50 μl reaction) 3. 2029 bp (GC content 74%) 4. 4988 bp (GC content 74%) β 1 gene 2005 bp (GC content 69%)
PrimeSTAR® Max DNA Polymerase PrimeSTAR® MAX DNA Polymerase PrimeSTAR® MAX DNA Polymerase
R045A R045B
Applications • • • •
Use Whenever Accuracy and Fidelity is Critical Cloning and Expression Studies Structure-Function Studies Analyses that involve Evolutionary Inferences (SNP analyses, evolutionary development experiments, etc.) • Whenever Fast PCR Cycles are Needed, such as High-Throughput Studies
Features • • • • •
Highest Fidelity: of any commercially available PCR polymerase Fastest Extension Speed: means less time required for PCR cycles Convenient Premix: assemble reactions in less time Antibody-Mediated Hot-Start Formulation Proven Performance: as reported in peer-reviewed literature
100 Rxns 400 Rxns
PCR. Since it is formulated as a premix that supports hot-start PCR, it's also excellent for high-throughput experiments. When you need fast reaction times and/or highly accurate amplification for cloning and expression, structural studies, or evolutionary analyses, PrimeSTAR Max DNA Polymerase is the enzyme of choice. PrimeSTAR Max DNA Polymerase is suitable for reactions occurring in the presence of excess nucleic acid. Such extraneous DNA in a reaction mix ordinarily inhibits PCR amplification when using conventional polymerases because the amount of effective polymerase available is limited by nonspecific binding. The superior processivity of PrimeSTAR Max DNA Polymerase prevents such inhibition by excess nucleic acid, resulting in a much higher success rate for PCR with minimal optimization of conditions required. Furthermore, the antibody-mediated hot start formulation prevents false initiation events during the reaction assembly due to mispriming and primer digestion. Since PrimeSTAR Max DNA Polymerase is configured as a 2-fold premix containing reaction buffer and dNTP mixture, it allows rapid preparation of reactions and is useful for high-throughput applications. PrimeSTAR® Max
Company T
Company S
M 1 2 3 4 5 6 7 8 M M 1 2 3 4 5 6 7 8 M 1 2 3 4 5 6 7 8
Description PrimeSTAR Max DNA Polymerase is a unique high-performance DNA polymerase for PCR. PrimeSTAR Max DNA Polymerase has the highest fidelity and fastest extension speed of any commercially available enzyme, along with extremely high sensitivity, processivity, and specificity. It includes an elongation factor to provide efficient priming and extension, greatly reducing the time required for annealing and extension steps. As a result, PrimeSTAR Max DNA Polymerase can be used for exceptionally fast high-speed
Good amplification was observed for products up to 6 kb using an extension time of 10 sec. with PrimeSTAR Max.
Clontech, A Takara Bio Inc. Company • www.clontech.com/takara
Template: λ DNA (1 ng/50 μl reaction) Target size: 0.5 kb (lane 1) , 1 kb (lane 2), 2 kb (lane 3), 4 kb (lane 4), 6 kb (lane 5), 8 kb (lane 6), 10 kb (lane 7), 12 kb (lane 8) Extension time: 10 sec
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9
High Fidelity PCR Reagents
to 30 kb (GXL). Simplify your PCR and save time by relying on one enzyme system works regardless of conditions, with minimal optimization required. PrimeSTAR GXL DNA Polymerase includes a modified PrimeSTAR HS enzyme and an additional elongation factor which in combination provide unsurpassed processivity. PrimeSTAR GXL DNA Polymerase has outstanding performance in reactions containing excess nucleic acid. Such extraneous DNA in a reaction mix ordinarily inhibits PCR amplification by conventional polymerases because the amount of effective polymerase available is limited by nonspecific binding. The superior processivity of PrimeSTAR GXL DNA Polymerase prevents such inhibition by excess nucleic acid, resulting in a much higher success rate for PCR with minimal optimization of conditions required. Furthermore, the antibody-mediated hot start formulation prevents false initiation events during the reaction assembly due to mispriming and primer digestion.
Applications
Takara Related Products Ordering Information
Takara Related Products
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Product Name Product No. Quantity Ligation T4 DNA Ligase 2011A 12,500 U (100 Weiss units) DNA Ligation Kit, Mighty Mix 6023 1 kit (75-150 rxns) DNA Ligation Kit, Version 2.1 6022 50 Rxns Cloning Vectors pBR322 DNA 3050 25 µg pUC18 DNA 3218 25 µg pUC19 DNA 3219 25 µg pUC118 DNA 3318 25 µg pUC119 DNA 3319 25 µg Protein Sequencing and Analysis Pfu Pyroglutamate Aminopeptidase 7334 10 mU Pfu Methionine Aminopeptidase 7335 20 mU Pfu Aminopeptidase 7336 0.5 mg Pfu N-acetyl Deblocking Aminopeptidase 7340 50 µg Arginylendopeptidase 7308 0.5 mg Asparaginylendopeptidase 7319 0.2 mU Endoproteinase Asp-N 7329 2 µg Pfu Protease S 7339 500 U Electrophoresis Mupid®-exU Electrophoresis System AD140 1 Unit Mupid®-2plus Electrophoresis System AD110 1 Unit Mupid® One Electrophoresis System AD160 1 Unit Power Supply for Mupid®-2Plus AD111 1 Unit Retroviral Transduction RetroNectin® Recombinant Human Fibronectin Fragment T100A 0.5 mg RetroNectin® Recombinant Human Fibronectin Fragment T100B 2.5 mg RetroNectin® Precoated Dish T110A 10 Dishes Ladders 1 kb DNA Ladder (Dye Plus) 3426A 100 Rxns 100 kb DNA Ladder (Dye Plus) 3422A 100 Rxns 20 bp DNA Ladder (Dye Plus) 3420A 100 Rxns 200 bp DNA Ladder (Dye Plus) 3423A 100 Rxns 250 bp DNA Ladder (Dye Plus) 3424A 100 Rxns 500 bp DNA Ladder (Dye Plus) 3425A 100 Rxns High Fidelity PCR Enzymes PrimeSTAR® GXL DNA Polymerase R050A 250 Units PrimeSTAR® GXL DNA Polymerase R050B 1000 Units PrimeSTAR® Max DNA Polymerase R045A 100 Rxns PrimeSTAR® Max DNA Polymerase R045B 400 Rxns Protease Inhibition Calpastatin 7316 3 mg
Takara has been manufacturing high quality restriction enzymes for over 30 years and offers more than 90 restriction enzymes to meet your cloning needs.
Visit our web site at www.clontech.com/takara to view these and other products. 30
Clontech, A Takara Bio Inc. Company • www.clontech.com/takara
Clontech Related Products Ordering Information Product Name Product No. Quantity TALON Prepacked HisTALON Gravity Columns 635655 5 columns HisTALON Gravity Columns Purification Kit 635654 20 purifications TALON Single Step Columns (20 ml) 635632 10 columns HisTALON Superflow Cartridges (5 x 1 ml) 635650 5 cartridges HisTALON Superflow Cartridge (1 x 5 ml) 635683 1 cartridge HisTALON Superflow Cartridges (5 x 5 ml) 635682 5 cartridges HisTALON Superflow Cartridge Purification Kit (5 x 1 ml) 635649 20 purifications HisTALON Superflow Cartridge Purification Kit (5 x 5 ml) 635681 5 purifications His60 Ni Superflow Resin His60 Ni Superflow 635659 10 ml 635660 25 ml 635661 4 x 25 ml 635662 250 ml 635663 2 x 250 ml 635664 4 x 250 ml His60 Ni Prepacked His60 Ni Gravity Columns 635657 5 columns His60 Ni Gravity Columns Purification Kit 635658 20 purifications His60 Ni Superflow Cartridges (5 x 1 ml) 635675 5 cartridges His60 Ni Superflow Cartridge (1 x 5 ml) 635680 1 cartridge His60 Ni Superflow Cartridges (5 x 5 ml) 635679 5 cartridges His60 Ni Superflow Cartridge Purification Kit (5 x 1 ml) 635674 20 purifications His60 Ni Superflow Cartridge Purification Kit (5 x 5 ml) 635678 5 purifications In-Fusion® HD Cloning System Liquid Kits In-Fusion® HD Cloning System 639645 10 Rxns 639646 50 Rxns 639647 100 Rxns 639692 96 Rxns In-Fusion® HD Cloning System CE 639636 10 Rxns 639637 50 Rxns 639638 100 Rxns 639693 96 Rxns Antibodies c-Myc Monoclonal Antibody 631206 200 μg HA-Tag Polyclonal Antibody 631207 100 μg c-Myc Monoclonal Antibody-Agarose Beads 631208 1 ml 6xHis mAb-HRP Conjugate 631210 100 μl 6xHis Monoclonal Antibody (Albumin-Free) 631212 200 μg 6xHN Polyclonal Antibody 631213 200 μl In-Fusion® HD Cloning System EcoDry™ Kits In-Fusion® HD EcoDry™ Cloning System 639684 8 Rxns 639685 24 Rxns 639686 48 Rxns 639688 96 Rxns Protease Inhibitor ProteoGuard EDTA-Free Protease Inhibitor Cocktail 635672 100 μL 635673 10 x 100 μL
Clontech Related Products
Clontech, A Takara Bio Inc. Company • www.clontech.com/takara
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Takara Product Offering Take advantage of Takara’s broad product portfolio and expert manufacturing capabilities to increase reliability and reproducibility, and to reduce your costs. Takara also offers custom, bulk and OEM services. Protein Research
Molecular Biology • Restriction and modifying enzymes • Cloning vectors • DNA/RNA ladders and MW markers • Electrophoresis salts and buffers PCR Polymerases and Reagents • Polymerases • Real-Time (qPCR) reagents • Reverse transcriptases • Primers and buffers
• Protease Inhibition • Protein Folding & Expression • Protein Sequencing and Analysis Glycobiology • Glycobiology Enzymes and Reagents • Glycobiology Kits Cellular Biology • Antibodies • EIA kits
Notice to Purchaser. Your use of these products and technologies is subject to compliance with any applicable licensing requirements described on the product’s web page at http://www.clontech.com/takara. It is your responsibility to review, understand and adhere to any restrictions imposed by such statements. Unless otherwise specified, other trade names are also the trademarks or registered trademarks of various companies.
TAKARA BIO INC.
TB 633353 Dist
www.clontech.com/takara
