Protein Expression and Purification 71 (2010) 28–32

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Expression and characterization of recombinant human retinol-binding protein in Pichia pastoris Monika Wysocka-Kapcinska, José Angel Campos-Sandoval, Akos Pal, John B.C. Findlay * The Marie Curie Laboratory for Membrane Proteins, National University of Ireland, Maynooth, County Kildare, Ireland

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Article history: Received 24 September 2009 and in revised form 13 January 2010 Available online 20 January 2010 Keywords: Pichia pastoris Human retinol-binding protein Transthyretin Recombinant protein Purification Fluorimetry

a b s t r a c t Plasma retinol-binding protein (RBP4) is the principal carrier of vitamin A in blood. Recent studies have suggested that RBP4 may have also a role in insulin resistance. To date the recombinant protein is usually produced by refolding inclusion bodies in Escherichia coli. Here we report the expression and characterization of recombinant human plasma RBP4 using the Pichia pastoris expression system. Simple and rapid purification allowed us to obtain 5 mg/L of purified protein from the fermentation supernatant with no need to perform denaturing and refolding steps. The identity of the protein was verified by ion-trap MS and Western blotting. The functionality of recombinant RBP4, i.e., the binding to its physiologic ligand, retinol, and the interaction with transthyretin (TTR), was tested by fluorimetric and pull-down assays, respectively. The apparent dissociation constant for retinol to the recombinant protein of 2  107 M was consistent with published data for native human protein. The recombinant protein interacted specifically with TTR. These results suggest that expression of recombinant human RBP4 in P. pastoris provides an efficient source of fully functional protein in soluble form for biochemical and biophysical studies. Ó 2010 Elsevier Inc. All rights reserved.

Introduction Plasma retinol-binding protein (RBP4),1 a monomeric protein with a molecular mass of 21 kDa, is the principal carrier of the essential vitamin A in the blood of vertebrates from its storage organ, the liver, to vitamin A-dependent tissues [1]. RBP4 circulates in the plasma associated with a second protein, the homotetramer transthyretin (TTR, 54 kDa) which is believed to increase the stability of the retinol–RBP4 complex and prevent its loss by glomerular filtration in the kidney [2]. The cellular uptake of retinol is mediated by a specific membrane receptor for RBP4 [3], now identified as STRA6 [4], with only the TTR-dissociated holo-RBP4 being able to bind to the receptor with high affinity [5]. Apo-RBP4, which shows a lower affinity for TTR than the holo form [6], is then excreted in the kidney owing to its small size [7]. Although the liver is the main organ for RBP4 synthesis, other tissues such as adipocytes also express this protein [8]. Recent work has suggested that RBP4 from adipose tissue may have a role in insulin resistance, obesity and ultimately type-II diabetes. RBP4 levels are elevated in these states and its normalization enhances insulin sensitivity.

* Corresponding author. Fax: +44 113 2333167. E-mail address: john.fi[email protected] (J.B.C. Findlay). 1 Abbreviations used: RBP4, retinol-binding protein 4; TTR, transthyretin; STRA6, stimulated by retinoic acid gene 6 homolog; SDS–PAGE, sodium dodecyl sulfate– polyacrylamide gel electrophoresis; BCA, bicinchoninic acid; MS, mass spectrometry; PBS, phosphate-buffered saline; YPD, yeast extract peptone dextrose; ECL, enhanced chemiluminescence. 1046-5928/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.pep.2010.01.015

Conversely, increased levels may contribute to insulin resistance [9–12]. Currently, human RBP4 has been purified from plasma or as a recombinant protein expressed in prokaryotic systems, but its affinity for TTR makes it difficult to purify from blood and the recombinant protein expressed in bacteria requires a denaturation and refolding process to be active [13]. Here we describe the expression of biologically active RBP4 as a soluble protein with a N-terminal His-tag that allows its efficient purification from fermentation supernatant using the eukaryotic expression system of Pichia pastoris. Materials and methods Strains and reagents Pichia pastoris strain KM71H (aox1::ARG4, arg4), expression vector pPICZ-a A and Zero Blunt TOPO PCR cloning kit were purchased from Invitrogen. Retinol, native human RBP and TTR, and cobalt affinity gel were from Sigma. Ni–NTA superflow resin was purchased from Qiagen. Anti-human RBP4 polyclonal antibody was from Dako. Other reagents were obtained from standard commercial sources. Construction of recombinant human RBP4 expression vector The coding sequence for mature human RBP4 (corresponding to residues 19–201) was originally cloned into the pQE30 vector [14].

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The resultant construct was used as a template DNA for PCR. The oligonucleotides designed to amplify the insert were: forward primer 50 -GAATTCCACCATCATCATCATCATGAGCGCGACTGCCGAGTGA GC-30 and reverse primer 50 -CTAAGCGTAATCTGGAACATCGTATG GGTACAAAAGGTTTCTTTCTGA-30 . The forward primer included the sequence for EcoRI restriction site (underlined) and a 6 Histag. The reverse primer included the sequence for the HA epitope and the stop codon. The PCR product was inserted into the pCRBlunt II TOPO vector, digested with EcoRI and introduced into the EcoRI sites of pPICZa A vector under the control of AOX1 promoter. Plasmid isolation and gel extraction were performed according to manufacturer’s instruction (Qiagen). The correct His-RBP4-HA/ pPICZa A construct was confirmed by digestion and automated sequencing. Transformation of P. pastoris and selection of transformants Pichia pastoris KM71H cells were transformed with the expression vector His-RBP4-HA–pPICZa A linearized by digestion with PmeI, using the lithium chloride method according to the P. pastoris expression manual (Invitrogen). The transformants were spread on YPD plates (1% yeast extract, 2% peptone (BD), 2% glucose, 2% agar) containing 100 lg/ml zeocin for growth and selection. After incubation at 30 °C for 48 h, large colonies were selected for subsequent analysis. Fermentation and time course expression study Expression of His-RBP4-HA was tested in randomly selected integrants. Transformants were grown on phosphate-buffered YP medium (1% yeast extract, 2% peptone, pH 7.5) in the presence of 2% glycerol (w/v) until the OD600 was between 2.0 and 6.0. The cells were harvested by centrifugation at 7000g for 5 min and used to inoculate 100 ml of YP medium to OD600 of 1.0 supplemented with 1% methanol (v/v) to induce expression. In each of 3 days, pure methanol was added to a final concentration of 1% (v/v). Aliquots of the cultures were collected every 24 h to monitor expression and secretion of RBP4. Purification of recombinant RBP4 The recombinant His-RBP4-HA was expressed in P. pastoris cells grown under optimized conditions. After 48 h of induction, the culture was centrifuged at 7000g for 5 min and the supernatant was collected. Subsequent steps were performed at 4 °C with protease inhibitors. The culture supernatant (4 L) containing recombinant RBP4 was incubated with Ni–NTA superflow resin. After incubation, the resin was packed to a disposable column (Pierce) and washed with PBS containing 10 mM imidazole to eliminate nonspecifically retained proteins. The bound proteins were eluted with elution buffer (PBS containing 250 mM imidazole) and the fractions analyzed by SDS–PAGE (15% gels) with silver or Coomassie staining. Those showing the presence of recombinant RBP4 were pooled and dialyzed against PBS buffer and then concentrated by ultrafiltration. The protein concentration was determined using the BCA assay with bovine serum albumin as standard. Western blot analysis Protein samples were separated on 15% SDS–PAGE gels and transferred to PVDF membranes. After blocking overnight at 4 °C with 10% (w/v) milk in PBST buffer (0.05% Tween 20 in PBS), membranes were incubated with rabbit anti-human RBP4 antibody (1:1000 dilution in PBST with 1% milk) for 2 h at room temperature. After 5 washes with PBST, the membrane was incubated with a secondary anti-rabbit IgG–horseradish peroxidase conjugate

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(1:5000 dilution in PBST) for 1 h at room temperature. Finally, the membrane was washed 3 times and the specific protein bands were visualized with the ECL system (Amersham). Prestained protein markers (broad range, 10–250 kDa) were used as a control for protein transfer and estimation of molecular mass.

Ion-trap MS of recombinant RBP4 Mass spectrometry (MS) of recombinant RBP4 was performed on an LC/MS system consisting of a 1200 Series liquid chromatograph, an HPLC-Chip Cube MS interface, and SL IT mass spectrometer (all Agilent Technologies, USA). The chromatographic chip incorporated a 40 nL enrichment column, a 150 mm/75 lm analytical column packed with Zorbax 300SB-C18 5 mm particles and a nanospray needle. In-gel protein digests were adapted from Shevchenko et al. [15].

Retinol binding assay The binding of retinol to His-RBP4-HA was monitored by fluorimetric titration in a Cary Eclipse fluorescence spectrophotometer (Varian). The purified recombinant protein was diluted to a concentration of 2 lM in PBS buffer and small increments of retinol solution in ethanol were added. The system was mixed and allowed to equilibrate for 5 min before recording the fluorescence emission of the retinol–RBP4 complex. The final concentration of ethanol never exceeded 2%. A solution of N-acetyl-L-tryptophanamide was used as a blank. For titration of RBP4 with retinol, excitation wavelength was 335 nm and emission was recorded at 470 nm. When binding of retinol was evaluated by following the quenching of protein fluorescence due to energy transfer to retinol, excitation wavelength was 280 nm and emission was recorded from 300 to 550 nm. The apparent dissociation constant (K 0d ) of retinol from RBP4 was estimated as described by Cogan et al. [16]. Briefly, a working equation was derived from the mass law equation in the convenient form:


P0 a ¼ ð1=nÞ½R0 a=ð1  aÞ  K 0d =n

ð1Þ

where R0 and P0 are the total retinol and protein concentrations, respectively, n is the number of independent sites for retinol and a is the fraction of free binding sites on the protein. a was calculated from fluorescence values by using the relationship:

a ¼ ðF max  FÞ=ðF max  F 0 Þ

ð2Þ

where F is the fluorescence intensity at a certain R0, Fmax is the fluorescence intensity upon saturation and F0 is the initial fluorescence intensity.

TTR interaction assay The binding of recombinant RBP4 to TTR was tested by pulldown assay. Purified recombinant His-RBP4-HA was incubated with native TTR for 1 h at room temperature in PBS. The HisRBP4-HA was pulled down by its His-tag using cobalt affinity gel. The beads were washed with PBS four times and suspended in SDS gel loading buffer. After 5 min boiling, the samples were analyzed by SDS–PAGE and Coomassie staining. The same pull-down experiment was carried out using native RBP4 and purified recombinant His-TTR to demonstrate that recombinant His-RBP4-HA behaves in an identical way to the native RBP4. A negative control experiment was carried using a different protein of similar size to TTR (lysozyme) to prove the specificity of RBP4:TTR interaction.

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Results

kDa

Expression of RBP4 from P. pastoris The coding sequence for recombinant human RBP4 (including His and HA tags) was placed in the pPICZa A vector under the control of the AOX1 promoter. This vector contains the a-factor prepro signal sequence from the yeast Saccharomyces cerevisiae in place of the original signal sequence allowing the recombinant protein to be secreted in the P. pastoris growth medium. The cells were transformed with linearized His-RBP4-HA/pPICZa A vector and plated on YPD medium containing zeocin. After 48 h, three independent, large colonies were induced by methanol in small cultures for 3 days, and supernatant and pellets were collected every 24 h for analysis by SDS–PAGE and Western blotting with specific anti-RBP4 antibodies. All three colonies had similar expression patterns. Secreted RBP4 in the growth medium could be observed as a band with an apparent molecular mass of 23 kDa (2.5 kDa corresponds to the His and HA tags). There was a progressive accumulation of RBP4 in the medium post induction that reached the maximum by 48 h (Fig. 1).

1

2

3

4

95 72 55 36 28

17

11

Fig. 2. Purification of His-RBP4-HA. SDS–PAGE analysis of purified His-RBP4-HA samples from P. pastoris. Lane 1, molecular mass markers; lane 2, culture supernatant before induction; lane 3, culture supernatant after 48 h of induction with methanol; lane 4, purified protein after elution from Ni–NTA resin with imidazole (200 ng). The gel was stained with silver.

Purification and identification of His-RBP4-HA The culture supernatant (4 L) was submitted to metal-chelate affinity chromatography on a Ni–NTA agarose column taking advantage of the N-terminal 6 His-tag in the recombinant protein. The bound RBP4 was eluted with 250 mM imidazole and the eluted fractions analyzed by SDS–PAGE and Coomassie or silver staining. As shown in Fig. 2, His-RBP4-HA was purified to homogeneity exhibiting one band with an apparent molecular mass of about 23 kDa, in agreement with the predicted Mr for the recombinant tagged protein (22.9 kDa). The amount of purified protein was 20 mg from a 4-L culture which contained 40 mg of secreted recombinant protein (50% recovery yield). The identity of the protein was confirmed by Western blotting analysis as described in previous section and by ion-trap MS. Mascot protein scores greater that 81 are significant (p < 0.05). After ingel digestion, we obtained a Mascot score of 358 with a sequence coverage of 73% and 8 human RBP4 peptides identified (matched peptides shown in bold, Fig. 3). Functional characterization of recombinant RBP4 The functionality of recombinant RBP4 was evaluated by testing its capacity to bind increasing concentrations of retinol. Upon

after methanol feed (h)

kDa

0

supernatant 72 24 48

0

pellet 72 48

E 48

excitation at 280 nm, the protein exhibited the typical fluorescence of the holo form: one peak with a maximum at 335 nm caused by the protein itself, and a second with a maximum at 470 nm, due to retinol bound to the protein (Fig. 4A). The retinol quenches the intrinsic RBP4 tryptophan fluorescence due to energy transfer to the vitamin resulting in an emission fluorescence peak at 470 nm (Fig. 4B). The data obtained were used to estimate an apparent dissociation constant for the retinol–RBP4 complex of 200 nM, in agreement with that reported in the literature for the protein isolated from human plasma [16–18]. Also, the ability of recombinant RBP4 to bind its partner TTR was evaluated by pull-down assay. Cobalt affinity gel was used in this assay since during the assay optimization we found less non-specifically bound TTR to the resin compared to Ni–NTA. As shown in Fig. 5, we detected specific interaction of the purified recombinant RBP4 with native TTR. Panel B shows a clear band in the third lane corresponding to the native TTR pulled down by its interaction with the recombinant His-RBP4-HA. To demonstrate that the recombinant RBP4 was behaving in an identical way to the native RBP4, we carried out the same pull-down assay using recombinant His-TTR and native RBP4 (panel C). In the third lane of panel C, the eluted fraction contained both proteins showing that they interacted and eluted together. No non-specific binding was detected, either to the resin (panel A), where no His-tagged protein was present, or to the recombinant RBP4 (panel D), where a different protein to TTR (lysozyme) was used.

N Discussion

28

17 Fig. 1. Time course of His-RBP4-HA expression. Western blot analysis with antihuman RBP4 antibodies of supernatant and cell pellet of His-RBP4-HA/pPICZa P. pastoris culture at different times (in hours) after the methanol induction. As a negative control, we analyzed the supernatant of pPICZa P. pastoris (E: empty vector) culture at 48 h after methanol induction. Native human RBP4 (N) was run as a positive control on the same gel.

The aim of the present work was to produce a large amount of a soluble and active recombinant human RBP4 suitable for biological studies without making use of denaturing agents or refolding steps during the purification procedure. Human RBP4 has been purified from plasma or urine, or produced as a recombinant protein expressed as inclusion bodies in Escherichia coli. In the first case, its affinity for TTR makes the purification process laborious, involving first a partial purification of the RBP4–TTR complex and then a separation of both proteins by various methods [1,17,18]. In the second, the recombinant RBP4 is expressed as inclusion bodies so requires solubilization and refolding steps in order to be active [13]. Here we report the expression and purification of soluble and biologically active RBP4 in the methylotrophic yeast P. pastoris.

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Fig. 3. Recombinant RBP4 identification by ion-trap MS. Matched peptides are shown in bold in the human RBP4 sequence.

A

B

RR NT

C

D

RR NR RT L

Fig. 4. Retinol binding assay. (A) Emission spectrum of His-RBP4-HA titrated with retinol. Excitation wavelength was 280 nm. Emission was recorded from 300 to 550 nm. The titration system consisted of 0.5 ml of 2 lM of the purified protein in PBS, pH 7.4. Ethanolic solutions of retinol were added to the cuvette until a final concentration of 3 lM. (B) Titration of apo-His-RBP4-HA with retinol as followed by the increase in fluorescence intensity. Excitation and emission wavelengths were 335 and 470 nm, respectively. Observed fluorescence intensity (s: RBP); fluorescence intensity of free retinol in N-acetyl-L-tryptophanamide solution (: TRY); fluorescence intensity corrected for fluorescence contribution of free retinol (d: RBP-TRY).

This eukaryotic system is well known for efficient secretion of heterologous proteins and is a superior alternative to E. coli expression. In this study, the human recombinant RBP4 was successfully expressed under the control of the alcohol oxidase (AOX1) methanol inducible promoter and was secreted into the medium through the cell wall using the S. cerevisiae a-factor signal sequence. This fact, combined with one-step metal-chelating affinity purification taking advantage of the His-tag, makes RBP4 purification very efficient in terms of time and yield: 5 mg of purified protein was obtained from the supernatant of 1 L of culture after 48 h of induction. HA epitope was included to give additional detection and purification options. We have also been able to use the P. pastoris expression system to produce recombinant RBP4 without any tags (data not shown) and other versions with different tags are in preparation. The expression and recovery levels can be further enhanced by adjusting culture conditions. The recombinant RBP4 showed the same properties as native human protein. Its functionality was examined by determining its affinity to bind retinol and to interact with TTR. The titration of recombinant RBP4 with small increments of retinol produced

Fig. 5. TTR binding assay. Purified His-RBP4-HA was evaluated by its capacity to interact specifically with TTR. His-RBP4-HA was incubated with native TTR and then pulled down with cobalt affinity resin which specifically binds the His-tag on RBP4. A parallel assay was included using native RBP4 and recombinant His-TTR. A different protein of similar size to TTR (lysozyme) was used as a negative control for non-specific binding. Bound proteins were analyzed by SDS–PAGE and Coomassie staining. Lanes represent flow-through (1), wash (2) and elution fractions (3) from the resin using (A) native TTR only, (B) recombinant His-RBP4-HA incubated with native TTR, (C) native RBP4 incubated with His-TTR and (D) recombinant His-RBP4HA incubated with lysozyme. The arrows indicate recombinant His-RBP4-HA (RR), native RBP4 (NR), recombinant His-TTR (RT), native TTR (NT) and lysozyme (L).

quenching of protein fluorescence by the vitamin and an enhancement of the retinol emission from which an apparent dissociation constant of about 200 nM was obtained, in agreement with the value previously published for the native protein [16]. Recombinant and native RBP4 showed identical binding affinity to TTR. We can demonstrate that His-RBP4-HA interacts with its membrane receptor (STRA6) in a retinol sensitive fashion using soluble membrane fraction from mammalian kidney cells naturally expressing STRA6, as already published [14,19]. In conclusion, we have developed a stable human RBP4 expression system in P. pastoris cells that will facilitate the production of large amounts of biologically active RBP4, convenient for further biochemical and biophysical studies. Acknowledgments This work was supported by the Marie Curie Transfer of Knowledge Scheme (MTKD-CT-2006-042480). We want to thank Dr. J.W. Kelly (The Scripps Research Institute, California, USA) for the TTR expression plasmid. References [1] M. Kanai, A. Raz, D.S. Goodman, Retinol-binding protein: the transport protein for vitamin A in human plasma, Journal of Clinical Investigation 47 (1968) 2025–2044.

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