Overview
Protein Expression Group
Protein Expression Group
Methods of protein expression with special respect to structural biology
Subunit B8 Human NADHUbiquinone Oxidoreductase Complex I
Bright/ARID domain from the human JARID1C
• Introduction • Techniques toward protein expression • Special demands for NMR-supported structural biology
human p47 SEP domain
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Anne Diehl // PhD Lecture FMP // 16.04.2008
Anne Diehl // PhD Lecture FMP // 16.04.2008
introduction
techniques
NMR-demands
introduction
Definition
techniques
Sugar-phosphate backbone
Protein Expression Group
Protein Expression Group
NMR-demands
DNA Structure
Lab slang: protein expression New combination of genetic material e.g. genes of human proteins combined with E.coli genome in an E.coli cell Example: Host: E.coli - biofactory Source: human
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• 1953 Crick and Wilkins, together with Watson, proposed the double-helix structure of DNA • (Nobel Prize 1962).
base pair
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introduction
techniques
NMR-demands
introduction
techniques
Codon usage
3 Nucleotids 1 Codon 1 Amino acid 61 codons 20 Amino acids
Protein Expression Group
Protein Expression Group
ATCG
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techniques
NMR-demands
introduction
techniques
Situation:
NMR-demands
Situation: Huge Genome projects running
scientific projects to determine the complete genome sequence of an organism
100 000s of targets available
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Protein Expression Group
Huge Genome projects running Protein Expression Group
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http://www.kazusa.or.jp/codon/
3 stop codons
introduction
NMR-demands
methods: result:
Databases with sequenz data free available
DNA isolation and sequencing complete order of 4 bases (ATCG) in a gene. determines which protein a gene will produce. http://www.genomesonline.org/
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introduction
techniques
NMR-demands
introduction
• Request material from authers of relevant publications • Material Transfer Agreement – Signed by head of the institute 9
introduction
techniques
NMR-demands
Protein Expression Group
Protein Expression Group
How to get the gene ? • Call a friend / cooperation partner
techniques
Order genes / clones / templates (natural sources)
http://www.imagenes-bio.de
NMR-demands
introduction
techniques
NMR-demands
Order genes / clones / templates (natural sources)
Protein Expression Group
Protein Expression Group
Order genes / clones / templates (natural sources)
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Berlin Buch
http://clones.invitrogen.com/
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http://www.genecopoeia.com/
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introduction
techniques
NMR-demands
introduction
techniques
NMR-demands
perfectly fitting for the host (adapted codon usage)
Or buy the protein of choise directly. Wide range of proteins available on the market!! extreme expensive!!
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www.geneart.com
introduction
Protein Expression Group
Protein Expression Group
Let the gene synthezise
techniques
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NMR-demands
introduction
techniques
NMR-demands
buying the Protein of interest? Protein Expression Group
Protein Expression Group
Taiwan
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• 100 µg ca. 500 € (functionell studies) • 10 mg ca. 50 000 € (structural studies) • the annual earning of two of you • Nevertheless screen the offers, catalogs, databases, not only publications – host, yields, constructs, competition situation
• get an idea, about your current market value or your value creation
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introduction
techniques
NMR-demands
introduction
Casting a host
• Introduction • Techniques toward protein expression • Special demands for NMR-supported structural biology
Anne Diehl // PhD Lecture FMP // 16.04.2008
introduction
techniques
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NMR-demands
introduction
IB
15 - 20% soluble / 20 - 40% inclusion bodies remainder do not express well or are degraded – inclusion bodies /incorrect folding – codon usage
• Solution
– fusion proteins (TRX / GST) / co-expression chaperones / lower temperature – plasmids for tRNA of rare codons RPIL / Rosetta 19 strains
techniques
NMR-demands
Codon usage Protein Expression Group
Protein Expression Group
Expression of human Proteins in E. coli
• Problems
NMR-demands
Protein Expression Group
Protein Expression Group
Overview
techniques
http://www.kazusa.or.jp/codon/
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L L L TSG
NMR-demands
L
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SLLL/L/LS/LL/L/S/LP/R/R
introduction
techniques
introduction
techniques
NMR-demands
Expression of human Proteins in E. coli
Protein Expression Group
R
techniques
http://faculty.ucr.edu/~mmaduro/codonusage/usage.htm
Protein Expression Group
introduction
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Rebecca Page
http://www.abrf.org/ResearchGroups/ProteinExpressionResearchGroup/Activities/Page-PERG2007.pdf
NMR-demands
introduction
techniques
NMR-demands
GOI-POI-DOI
Protein Expression Group
Protein Expression Group
gene-protein-domain of interest
Expression test STAS-domain (B. subtilis) //SDS-PAGE Rosetta2
Gold pLysS 23
• NMR structural biology – size limit 20 kDa • Full length proteins often too large, signal overlap, bad resolution • Targets Domains
– Recognition by bioinformatic tools like SMART & PFAM Peripheral plasma membrane protein CASK (1000AA)
– Cloning a domain with variable domain bounderies – secondary structure prediction 24
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introduction
techniques
NMR-demands
introduction
Cloning
Bioinformatics
Bam HI (5238)
Ava I (5208)
Nco I (5225)
Xma I (5208)
LIC site
Protein Expression Group
NMR-demands
Vector / GOI / Recombination
Ava I (5278)
T7 terminator
His tag
f1 origin
enterokinase S tag thrombin
kan sequence
His tag
Xma I (1068)
T7 promoter lac operator ClaI (4968) Apa LI (4263)
– carries a circular chromosome – may carry a circular extra-chromosomal genetic information called vector or plasmid easily introduced into cells multiple copies per cell
Ava I (1068) Sma I (1070)
pET-30 Ek/LIC rc
ClaI (1251)
5439 bp
lac I Apa LI (1828)
ColE1 pBR322 origin Apa LI (2328)
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VectorNTI free for academic user at Invitrogen
introduction
techniques
NMR-demands
Vector types Protein Expression Group
techniques
Sma I (5210)
• Easiest host: E. coli
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Cloning Vectors HindIII (5263) Eco RI (5244)
Protein Expression Group
– reproducing the gene – production of the coded protein
• Secondary structure Jpred – http://www.compbio.dundee.ac.uk/~www-jpred/ • Domain Smart/Pfam – http://smart.embl-heidelberg.de/ – http://www.sanger.ac.uk/Software/Pfam/ • Disorder Prediction DisEmbl – http://dis.embl.de/ • 3D structures PDB – http://www.rcsb.org/pdb/ • Refolding database – http://refold.med.monash.edu.au/ • Target DB
– http://targetdb.pdb.org/
NMR-demands
Vector / GOI / Recombination
• Put a gene in a vehicle /vector for
https://catalog.invitrogen.com/index.cfm?fuseaction=userGroup.userGroupHome
Protein Expression Group
all tools easy to use: paste your AA sequence
introduction
techniques
• T5 promotor e.g. pQE, pGEX
– E.coli RNA polymerse is compatible – Expression in all strains DH5alpha, SCS1, JM109 etc. – Expression leaky, not suitable for toxic proteins
• T7 promotor e.g. pET – Needs T7-RNA-polymerase (DE3) – E.coli (DE3) only suitable!!
T7 and T5 stand for virus types
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introduction
techniques
NMR-demands
introduction
techniques
NMR-demands
Vector / GOI / Recombination
Restriction endonucleases
• • • •
Expression of target gene is repressed at multiple levels in pET system vectors Lac repressor controls expression of T7 RNA polymerase (exogenous, from T7 phage), target vector is under control of T7 promoter Lac repressor also controls expression of target gene directly, in case small amounts of T7 polymerase are present prior to induction with IPTG T7 lysozyme inhibits T7 polymerase in cells containing pLysS or pLysE plasmid29
introduction
techniques
Protein Expression Group
Protein Expression Group
• Enzymes from bacterial cells
NMR-demands
Bam HI
Bacillus amyloliquefaciens
G GATCC
Eco RI
Eschericia coli RY13
G AATTC
Hind III
Haemophilus influenza Rd
A AGCTT
Nae I
Nocardia aerocolonigenes
GCC GGC
Pst I
Providencia stuartii
CTGCA G
Sma I
Serratia marcescens
CCC GGG 31
techniques
NMR-demands
Vector / GOI / Recombination
Polymerase Chain Reaction
Protein Expression Group
Protein Expression Group
Recognition
– like HANNAH OTTO REGAL LAGER
• Nobel prize 1978 (Arber, Nathans, Smith)
introduction
Restriction endonucleases Microorganism source
• Used to "cut and splice" DNA • Recognise specific DNA sequences restriction sites / usually 4-8 bp • Always cut DNA at the same site • most sites are palindromic
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Vector / GOI / Recombination
Name
– Protective mechanism – Degrade DNA of invading viruses
1. Separate both DNA-strands by heating (denature the DNA). 2. Cool slightly. 3. Build new strand from primers (short /ca. 20 bp / single stranded DNA pieces) and nucleotides by a heat stabile enzyme (Taq Thermus thermophilus /aquaricus) 4. Repeat 25-30 times. 95ºC Denature
Extend primers
72ºCMg++ Primer , dNTP, Taq polymerase, Anneal primers
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50-60ºC
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techniques
NMR-demands
Protein Expression Group
PCR - principle gt
g
c taa
ac tta
Round 1
introduction
Vector / GOI / Recombination
c tat ga
caattg gttaac
Round 2
- the overhang encode a new restriction site, - corresponding to the restriction sites of the chosen vector
aattg c
g ctata
Unidirectional insertion Sub-clone in vector
atat ttaa Promoter Antibiotic resistence
NMR-demands
Critical steps Digest of PCR products
introduction
Vector / GOI / Recombination
Cut preferred in the middle of a DNA fragment Primer design: NNNNNN-REgene specific bases LIC Ligation independ cloning35
Protein Expression Group
Endonucleases
techniques
MCS multiple cloning site ca. 15 restriction sites Preparation with the same Enzymes used for PCR— product digest
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NMR-demands
Vector / GOI / Recombination
LIC qualified PCR products
Ligation
Restriction
Protein Expression Group
Vector / GOI / Recombination
gatatc ctatag
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techniques
NMR-demands
Restriction digestion
c tat ga
caattg gatatc gttaac ctatag Restriction site addition to the gene of interest via primer design and amplification
introduction
techniques
PCR-product
Protein Expression Group
introduction
two different 14 bp sticky ends can be inserted simultanously in all available vectors annealed products ready for transformation in E.coli/ Insects etc. 36
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introduction
techniques
NMR-demands
introduction
Vector / GOI / Recombination
Advantages of LIC
techniques
NMR-demands
Protein expression is easy Sma I (5969) Ava I (5967) Xma I (5967)
TR8
Hin dIII (6049)
Ava I (6064)
BamHI (5997) NcoI (5984) EcoRI (6003) Pst I (6032)
His Tag T7 terminator f1 origin
LIC site enterokinase
Kan coding sequence
S Tag
XmaI (1068)
thrombin Sma I (5969)
HindIII (6049) Ava I (5967) Bam HI (5997) Nco I (5984) EcoRI (6003) XmaI (5967) Pst I (6032)
His Tag
GST Tag Sma I (5969)
Hinpromoter dIII (6049) T7 BamHI (5997) Ava I (5967) lac operator
T7 terminator f1 origin
NcoI (5984) (6003) Xma I (5967) ClaEcoRI IPst (4731) I (6032)
XmaI (1068) enterokinase
S Tag AvaI (1068) thrombin
Hin dIII (6049)
lac operator NcoI (5984)
2. Low Background: Reported to be
