Expression and Purification of Cytokine Receptor Homology Domain of Human Granulocyte-Colony-Stimulating Factor Receptor Fusion Protein in Escherichia coli

Protein Expression and Purification 21, 87–91 (2001) doi:10.1006/prep.2000.1343, available online at http://www.idealibrary.com on

Expression and Purification of Cytokine Receptor Homology Domain of Human Granulocyte-ColonyStimulating Factor Receptor Fusion Protein in Escherichia coli Daisuke Tatsuda, Haruhiko Arimura, Hiroko Tokunaga, Matsujiro Ishibashi, Tsutomu Arakawa,* and Masao Tokunaga 1 Laboratory of Applied and Molecular Microbiology, Faculty of Agriculture, Kagoshima University, 1-21-24 Korimoto, Kagoshima 890-0065, Japan; and *Alliance Protein Laboratories, 3957 Corte Cancion, Thousand Oaks, California 91360

Received June 16, 2000, and in revised form August 21, 2000

Direct expression of the cytokine receptor homology (CRH) domain of granulocyte-colony-stimulating factor (G-CSF) receptor is lethal to Escherichia coli. For the efficient and stable production of an active CRH domain in E. coli, we fused the CRH domain with different proteins, such as maltose-binding protein (MalE), glutathione S-transferase, and thioredoxin (Trx). Among these, Trx appeared to be the best in terms of the protein expression level, purification efficiency by affinity chromatography, and binding activity to its ligand, G-CSF. The yield of active Trx–CRH fusion protein increased about 200-fold compared to that of previously reported MalE–CRH fusion. © 2001 Academic Press

Granulocyte-colony-stimulating factor (G-CSF) 2 is one of the hemopoietic growth factors and is administered as an important therapeutic agent (1, 2). Several derivatives of recombinant human G-CSF and a nonpeptidyl mimic of murine G-CSF have been developed (3, 4). An efficient expression system for active G-CSF receptor molecule is urgently needed in order to screen more potent derivatives and mimetics of G-CSF and determine the high resolution three-dimensional structure of the receptor/G-CSF complex. The extracellular domain of the G-CSF receptor consists of an immunoglobulin-like domain, a cytokine 1 To whom correspondence should be addressed. Fax: 81-99-2858634. E-mail: [email protected]. 2 Abbreviations used: G-CSF, granulocyte-colony-stimulating factor; CRH, cytokine receptor homology; MalE, maltose-binding protein; GST, glutathione S-transferase; Trx, thioredoxin; PBS, phosphate-buffered saline; SDS–PAGE, SDS–polyacrylamide gel electrophoresis.

1046-5928/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.

receptor homology (CRH) domain, and fibronectin type III repeating domains (5, 6). The CRH domain is believed to be essential for binding to G-CSF (7). Previously, we attempted to produce a nonglycosylated CRH domain of the human G-CSF receptor in Escherichia coli and succeeded in direct expression of the CRH domain with a carboxy-terminal His tag (8). However, direct expression of the CRH domain of G-CSF receptor was toxic to E. coli resulting in low yields (8). Protein fusion techniques are powerful for the expression of heterologous proteins in E. coli (9). Fusion proteins are generally easily identified and can be highly purified using the unique affinity of the partner protein for its ligand. The fusion partner appears to facilitate the folding of fusion proteins and serves as an affinity tag and as a spacer to conjugate fusion protein to the solid phase. We constructed CRH domain fusion protein with different fusion partners, maltose-binding protein (MalE), glutathione S-transferase (GST), and thioredoxin (Trx), for efficient and stable production of active CRH domain in E. coli. In the present paper, we report that Trx fusion yields the highest recovery of active CRH protein. MATERIALS AND METHODS

Medium and Strains E. coli strains JM109 and BL21(DE3) were used, and LB containing 100 ␮g/ml of ampicillin was used for the selection of transformants. Site-Directed Mutagenesis and Plasmid Construction The Cys 163 (tgc) and Cys 228 (tgc) residues of CRH domain have been changed to serine (tcc) residues by PCR87

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mediated site-directed mutagenesis (10) as follows. Mutated forward (5⬘-GGCAGAGCCACTGCTCCATCCCACGCAAAC-3⬘) and backward (5⬘-GTTTGCGTGGGATGGAGCAGTGGCTCTGCC-3⬘) primers for Cys 163 mutagenesis and mutated forward (5⬘-GCTGCCTACAGCTGTCCTGGGAGCCATGGC-3⬘) and backward (5⬘-GCCATGGCTCCCAGGACAGCTGTAGGCAGC-3⬘) primers for Cys228 mutagenesis were used. After mutagenesis, the region encoding the mutated CRH domain (Tyr 97 to Ala 309) was amplified by PCR using a forward primer (5⬘-CTGGTTCCGCGTGGATCCTACCCTCCAGCCATACCCCAC-3⬘), which contains a thrombin cleavage site followed by the coding sequence starting at Tyr 97 , and a backward primer (5⬘-CCGAATTCGGCCCGTTCGGTAGTTCTCAGC-3⬘), which contains the EcoRI site and coding sequence up to Ala309. The amplified fragment was digested with EcoRI and then ligated into XmnI/EcoRI-digested pMalp2 (New England BioLabs.) to construct plasmid pMal-R (Fig. 1). The DNA fragment encoding CRH domain was cut out by BamHI/ EcoRI and ligated to the BamHI/EcoRI-digested pGEX4T-1 (Pharmacia) to construct pGST-R (Fig. 1). The BamHI/XhoI fragment of pGST-R encoding CRH domain was ligated to the BamHI/XhoI-digested pET32a (Novagen), thioredoxin fusion vector, to construct pG-SDM-1 (Fig. 1). The DNA sequence was verified by the dideoxychain termination method described by Sanger et al. (11) using a Thermo sequenase cycle sequencing kit (Amersham US78500). Expression of Fusion Proteins E. coli JM109 cells harboring pMal-R or pGST-R were grown in LB/ampicillin at 37°C to A 600 ⫽ 1.0, and expression of the maltose-binding protein–CRH domain or glutathione S-transferase–CRH domain fusion proteins was induced by the addition of isopropylthiogalactopyranoside to give a final concentration of 0.1 mM. To express the thioredoxin–CRH domain fusion protein, BL21(DE3) cells harboring pG-SDM-1 were treated as above. After 2 h of induction, cells were collected by centrifugation at 7000 rpm for 10 min, suspended in phosphate-buffered saline (PBS, 20 mM sodium phosphate buffer, pH7.5, containing 0.14 M NaCl) and 1 mM phenylmethylsulfonyl fluoride, and disrupted by sonic oscillation with 50% pulse at dial 3 for a total of 5 min (Branson cell disruptor 200 with 21-in. tip). The crude homogenate was centrifuged at 14,000 rpm for 10 min to obtain soluble supernatant and insoluble pellet fractions. Purification of Fusion Proteins by Affinity Column Chromatographies All affinity columns were prepared in a Poly-Prep column (Bio-Rad, 731-1550) using 2-ml affinity resins. Soluble supernatant fraction of cell homogenate con-

taining MalE–CRH fusion protein was applied to an amylose column (New England BioLabs) equilibrated with 20 mM Tris buffer, pH 7.5, containing 200 mM NaCl and 1 mM EDTA. After washing with 6 column vol of the above buffer, the bound fraction was eluted with 20 mM Tris buffer, pH 7.5, containing 200 mM NaCl, 1mM EDTA, and 10 mM maltose. The insoluble MalE–CRH and the GST–CRH fusion proteins were solubilized with 50 mM Tris buffer, pH 8.0, containing 1 mM dithiothreitol and 8 M urea, centrifuged to remove cell debris, and dialyzed against 1 liter of 20 mM sodium phosphate buffer, pH 7.2, and 0.1 M sodium carbonate, pH 8.0, respectively, to remove the urea. After centrifugation at 14,000 rpm for 10 min, solubilized proteins were applied to each affinity columns. The GST–CRH fusion protein was eluted from a glutathione affinity column with Tris buffer, pH 8.0, containing 5 mM reduced glutathione. The thioredoxin–CRH fusion protein in insoluble pellet fraction was dissolved in 0.1 M sodium phosphate and 10 mM Tris–HCl buffer, pH 8.0 (P-buffer), containing 6 M guanidinium–HCl and was applied to Ni–NTA agarose column after centrifugation at 14,000 rpm. Column was washed with P-buffer, pH 8.0, containing 8 M urea. The Trx–CRH fusion protein was eluted successively with P buffer containing 8 M urea at pH 6.3, 5.9, and 4.5. G-CSF Affinity Column Chromatography G-CSF affinity column was prepared by coupling recombinant human G-CSF to CNBr-activated Sepharose 4B (13). The column was equilibrated with PBS, and the above three purified fusion proteins were applied to the G-CSF–Sepharose 4B column to evaluate their binding activities. Other Methods SDS–polyacrylamide gel electrophoresis (SDS– PAGE) was carried out according to Laemmli (12). Western blotting and immunostaining with anti-human G-CSF receptor antiserum (13) were described before (14). The amount of protein was measured according to Lowry et al. (15). The amount of protein in the band stained with Coomassie blue after SDS–PAGE was also measured by densitometry or using NIH Image software with bovine serum albumin as the standard. RESULTS AND DISCUSSION

Expression and Purification of MalE–CRH, GST–CRH, and Trx–CRH Fusion Proteins To reduce the possibility of incorrect disulfide bond formation due to the presence of free cysteines, we changed Cys 163 and Cys 228 to serine (tcc) by site-directed mutagenesis. Haniu et al. (16) have reported

Trx–CRH FUSION OF G-CSF RECEPTOR

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FIG. 1. Structures of expression vectors. pMal-R for MalE–CRH, pGST-R for GST–CRH, and pG-SDM-1 for Trx–CRH fusion proteins were constructed (see Materials and Methods). ptac, tac promoter; pT7, T7 promoter; Amp, ampicillin resistant.

that Cys 163 and Cys 228 of human G-CSF receptor are not involved in disulfide bond formation and that blocking the free cysteines does not affect the ligand binding. These two free cysteines in human G-CSF receptor are not conserved between different species and are replaced with serine residues in the mouse G-CSF receptor (6). A variety of systems for the expression of heterologous proteins fused to the affinity partner in E. coli have been described (9). We constructed MalE–CRH, GST–CRH, and Trx–CRH fusion proteins to improve the yield of active CRH domain and compared their characteristics. Trx was developed as a novel gene fusion partner with high expression, high solubility, and unique properties suitable for protein purification (17). Here, all expression and purification experiments were carried out at least four times and average deviation of the data was within 10%. Three fusion vectors, pMal-R, pGST-R, and pGSDM-1 (Fig. 1) were transformed into E. coli to characterize the expressed fusion proteins. All three transformants grew normally after induction of fusion proteins, suggesting that the CRH domain in fusion proteins is not toxic to E. coli. In crude homogenates, the protein band corresponding to molecular mass of Trx–CRH (Fig. 2A-1, lane 1), GST–CRH (Fig.2A-1, lane 2), and MalE–CRH (Fig. 2A-1, lane 3) were detected by Coomassie blue staining and by immunostaining with anti-G-CSF receptor antiserum (Fig. 2A2). The total amount of expressed Trx–CRH, GST– CRH, and MalE–CRH fusion proteins was 4.68, 1.12, and 0.71 mg per 50-ml culture, respectively. The molar ratios of CRH portion in total expressed fusion proteins were 13.8:2.3:1 for Trx–CRH:GST–CRH:MalE-CRH, demonstrating that the Trx fusion system is superior to the other two fusion systems for the expression of CRH domain (Table 1). Subcellular localization of these fusion proteins were determined: 89% of Trx– CRH, 100% of GST–CRH, and 65% of MalE–CRH were

localized in insoluble fractions of crude homogenate (Figs. 2A-1 and 2A-2, lanes 4 –9), indicating that the CRH domain was recovered mainly in insoluble fraction as nonnative protein for all three fusion proteins. Three fusion proteins were purified to apparent homogeneity by the respective affinity column chromatographies (Fig. 2B) after solubilization with 8 M urea or 6 M guanidinium–HCl followed by their removal. The yields of affinity purification are 40.5% (1.89 mg), 11.3% (0.126 mg), and 27.0% (0.192 mg) for Trx, GST, and MalE fusion proteins. This yield represented the amount of fusion proteins purified as a soluble form. Binding Assay of CRH Fusion Proteins Using G-CSF Affinity Column We studied binding of the CRH fusion proteins to a G-CSF affinity column to estimate refolding efficiency. As shown in Fig. 3, 190 ␮g of active Trx–CRH fusion protein, corresponding to 5.37 nmol of the CRH domain, was obtained from 50 ml of culture. This yield was about 200 times higher than that obtained with the MalE fusion (0.026 nmol of CRH domain, Table 1) which was used as a fusion partner for the expression of murine G-CSF receptor in the previous paper (18). The yield of active CRH domain in GST–CRH fusion was 0.104 nmol. Thus using a Trx fusion system, we succeeded in purifying a substantial amount of active CRH fusion protein for screening of G-CSF derivatives or small molecule mimetics. When heterologous proteins expressed in E. coli contain disulfide bonds and free cysteines, their production in the active form requires correct disulfide formation after cell disruption and purification (19). The CRH domain of human G-CSF receptor contains five disulfide bonds and it is hence expected that fusion partner proteins might profoundly affect folding efficiency of the CRH part of the fusion proteins. From our results, the Trx fusion system yields the

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FIG. 2. Expression and purification of fusion proteins. (A) Coomassie blue stain (A-1) and immunostain (A-2) of crude homogenates. Lanes 1, 4, and 5, BL21(DE3, pG-SDM-1); lanes 2, 6, and 7, JM109(pGST-R); lanes 3, 8, and 9, JM109(pMal-R). Lanes 1–3, crude homogenates; lanes 4, 6, and 8, supernatant fraction of crude homogenate; lanes 5, 7, and 9, pellet fraction of crude homogenate. MW, molecular weight marker. (B) Coomassie blue stain (B-1) and immunostain (B-2) of affinity-purified fusion proteins. Lane 1, Trx–CRH (T); lane 2, GST–CRH (G); lane 3, MalE–CRH (M). MW, molecular weight marker.

best results in terms of the total amount of expressed protein, purification efficiency by affinity chromatography, and binding activity to G-CSF. It is possible, however, that Trx fusion and other fusions may give a higher yield than described here after optimization of

expression and refolding. Refolding may be improved by adopting protocols used to refold cytokines, growth factors, or other insoluble proteins. Such an effort may be hampered for MalE and GST fusion proteins, since they tend to form aggregates by putative nonspecific

TABLE 1 Yields of CRH Fusion Proteins

Fusion proteins MalE fusion protein GST fusion protein Trx fusion protein

Amount expressed (␮g/50 ml) [molar ratio]

Expressed in insoluble fraction (%)

Yield of affinity purified protein (␮g/50 ml)

(␮g/50 ml)

710 [1.0] 1120 [2.3] 4680 [13.8]

65

84 (Sol.) 108 (Insol.) 126

0.8 (Sol.) 1.1 (Insol.) 6.3

100 89

1897

Yield of active fusion protein

190

[molar ratio] [1.0] [4.0] [204.3]

Note. Protein amounts (␮g/50 ml culture) were shown. Molar ratio, the ratio of mole amount of CRH domain in each fusion proteins, representing mole amount of MalE–CRH as one. Sol, protein from soluble fraction; Insol, protein from insoluble pellet fraction. Experiments were carried out at least four times and the average value is shown in this table. The deviation of the data was within 10%.

Trx–CRH FUSION OF G-CSF RECEPTOR

FIG. 3. Binding of CRH fusion proteins on G-CSF column. Each purified fusion protein (0.4 nmol of CRH domain) was applied to G-CSF affinity column. MW, molecular weight marker; F, flowthrough fraction of G-CSF column; lanes wash 1–9, washed fractions with buffer; lanes elution 1–3, eluted fraction with glycine buffer(pH 3.0)/2 M urea.

hydrophobic interaction and/or incorrect intermolecular disulfide bonds during solubilization of proteins from inclusion bodies. The presence of sulfhydryl reagents often yields higher binding of these fusions to the respective affinity column than does the absence of the reagents (data not shown). Therefore, Trx fusion may be a choice among various fusions to obtain a large quantity of active CRH domain of G-CSF receptor. Finally, higher binding activity to the ligand was observed for the Trx fusion than for other fusions. This suggests that Trx might facilitate the proper folding of the CRH domain or simply that the smaller fusion partner might exhibit less steric hindrance to the binding on the ligand molecules. The cysteine residues of Trx might function as a disulfide reshuffling agent for correct disulfide formation. In conclusion, this protein fusion approach improved the yield of the active CRH domain of human G-CSF receptor compared to that of the previous direct expression (8). We found that Trx is the best fusion partner for expression of CRH domain and succeeded in the simple purification of active fusion protein ready for use in screening potent G-CSF mimetics and for the study of CRH domain/G-CSF complex. REFERENCES 1. Bronchud, M. H., Scarffe, J. H., Thatcher, N., Crowther, D., Souza, L. M., Alton, N. K., Testa, N. G., and Dexter, T. M. (1987) Phase I/II study of recombinant human granulocyte colony-stimulating factor in patients receiving intensive chemotherapy for small cell lung cancer. Br. J. Cancer 56, 809 – 813.

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