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High-level expression, purification, and characterization of recombinant human basic fibroblast growth factor in Pichia pastoris
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Protein Expression and Purification 59 (2008) 282–288 www.elsevier.com/locate/yprep
High-level expression, purification, and characterization of recombinant human basic fibroblast growth factor in Pichia pastoris Xupeng Mu, Ning Kong, Weili Chen, Ting Zhang, Mohan Shen, Weiqun Yan * Department of Biochemistry, Institute of Frontier Medical Sciences, Jilin University, Xinmindajie Street 1163, Changchun, Jilin 130021, China Received 11 November 2007, and in revised form 14 Feburary 2008 Available online 29 February 2008
Abstract Basic fibroblast growth factor [basic FGF (bFGF); FGF-2] is an important member of the FGF family, bFGF is a potent angio genic molecule in vivo and in vitro stimulate smooth muscle cell growth, wound healing, and tissue repair. The full-length hbFGF coding sequence, gained by RT-PCR, was cloned into the pPICZaA vector in frame with the yeast a-factor secretion signal under the tran scriptional control of the AOX promoter and integrated into Pichia pastoris strain X33, and the high level expression of rhbFGF has been achieved. SDS–PAGE and Western blotting assays of culture broth from a methanol-induced expression strain demonstrated that rhbFGF, an 18 kDa protein, was secreted into the culture medium. The growth conditions of the transformant strain were optimized in 50 ml conical tubes including methanol concentration, pH and inducing time. Under the optimal conditions, stable production of rhbFGF around 150 mg/l was achieved. The expressed rhbFGF was purified more than 94% purity using SP Sepharose ion exchange chromatogra phy and source™ 30RPC. A preliminary biochemical characterization of purified rhbFGF was performed by biological activity analysis which was used by NIH/3T3 cell cultures, and the results demonstrated that the purified rhbFGF stimulated the growth of NIH/3T3 cells similarly to standard material. © 2008 Elsevier Inc. All rights reserved. Keywords: Basic fibroblast growth factor; Pichia pastoris; Secretory expression; Protein purification
The fibroblast growth factor (FGF)1 protein family of heparin-binding proteins currently consists of 22 structur ally-related polypeptides with similar biological activities. These include the two prototypes, FGF1 (aFGF) and FGF2 (bFGF) [1]. As a single-chain protein, bFGF has 146 amino acids and pI of 9.6, and Mr is about 17,200. Natural bFGF was isolated initially from pituitary extracts. BFGF has pleio tropic eVects in diVerent cells and organ systems. BFGF can stimulate smooth muscle cell growth, wound healing, and tissue repair [2,3]. In addition, bFGF may stimulate hemato poiesis [4] and may play an important role in the diVerentia tion and/or function of the nervous system [5,6], the eye [7], * Corresponding author. E-mail address: [email protected] (W. Yan). 1 Abbreviations used: bFGF, basic fibroblast growth factor; PBS, phos phate-buVered saline; OPD, ortho-phenylenediamine; DAB, 3,39-diam inobenzidine; FCS, fetal calf serum; BSA, bovine serum albumin; DMEM, Dulbecco’s modified Eagle’s medium; MTT, methylthiazoletetrazolium; DMSO, dimethyl sulfoxide. 1046-5928/$ - see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.pep.2008.02.009
and the skeleton [8]. In animal study, bFGF can prevent the brain from injury of ischemia and reperfusion in rats [9]. Therefore, obtaining large quantities of recombinant bFGF is important for both clinical studies and mechanistic investigations. But it was almost impossible to obtain suY cient bFGF from animal tissues due to its extremely low quantity and high expense. Therefore, in the last decade, genetic technology has been employed to produce bFGF at low cost. So far, recombinant bFGF which was used in clinic has been produced by Escherichia coli [10], the expres sion of rhbFGF by Pichia pastoris has not been reported yet. P. pastoris is a widely used, eYcient expression system for a wide variety of molecules [11]. P. pastoris is physically robust and amenable to high-density fermentation as E. coli but possess the necessary cellular machinery to carry out post-translational modifications. With the advantages of both prokaryotic and eukaryotic systems, P. pastoris provides the potential for producing soluble, correctly folded recombinant proteins that have
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undergone all the post-translational modifications required for functionality and is easy to handle and its fermentation conditions are very simple. Additionally, isolation of foreign protein is easy, because P. pastoris does not secrete a large amount of intrinsic protein [12]. In the present study, we have cloned the cDNA encoding hbFGF and achieved its high level secreting expression with a yield of 150 mg/l of yeast culture. Activity assay showed that rhbFGF had the biological function as the standard material. Materials and methods Cell, vector, host strains, and reagents Plasmids were amplified in E. coli XL1-Blue (Dingguo, China). P. pastoris and the P. pastoris integrative expression vector (pPICZaA) were obtained from Invitrogen (USA) and pPICZaA vector was reconstructed by our lab which did not have the two Ste13 cleavage sites. All media for growth of P. pastoris were prepared as the protocols obtained from man ufacturer (Invitrogen, USA). Restriction enzymes and T4 DNA ligase were purchased from Takara (Dalian, China). PCR purification kit, gel extraction kit, and Miniprep kit for plasmid extraction were obtained from Qiangen Company (USA). Primer was synthesized by Shenggong Company (Shanghai, China). Standard hbFGF was purchased from national institute for the control of pharmaceutical and bio logical products (China).
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Electroporation of X33 and screening for recombinant strain Plasmid DNA was linearized with SacI and was purified by phenol–chloroform extraction and ethanol precipitation. The purified DNA fragment, about 10 lg, was dissolved in 10 ll of ddH2O. Electrocompetent cells of P. pastoris X33 were prepared according to the supplier’s instruction [13]. Then 80 ll of competent cells were mixed with 10 lg line arized recombinant plasmid in a 0.2 cm electroporation cuvette. The mixtures were incubated on ice for 5 min, and the electroporation was carried out on GenePulser appara tus (Bio-Rad, USA) with the following settings: 1.5 kV volt age, 25 lF capacitance, and 200 X resistance. After pulsing, 1.0 ml 1 M ice-cold sorbitol was added immediately to the cuvette. The cells were transferred into a 1.5 ml sterile tube and incubated at 30 °C without shaking for 1 h. Then the transformed cells were plated on YPDS containing zeocin for 100 lg/ml and incubated at 30 °C for at least 3 days. Genomic DNA analysis To verify the recombinant gene integration, the g enomic DNA of a number of transformant recombinant P. pastoris strains were detected by PCR assay. The following primers were used: AOX1 universal primers (sense: 59-G ACTGGTTCCAATTGACAAGC-39, anti-sense: 59-GC AAATGGCATTCTGACATCC-39). Thirty cycles of ampli fication were carried out at 94 °C for 30 s, 55 °C for 30 s, and 72 °C for 1 min.
RNA extraction and RT-PCR Induced expression of rhbFGF in P. pastoris Total RNA was extracted from human glioma cells and used as the template for RT-PCR. The primers were 59-ATACTCGAGAAGAGAGCAGCCGGGAGCATCA CCA-39 (forward primer), which contains an XhoI site (underlined) and the kex2 site (underlined), and 59-GCGGA ATTCTCAGCTCTTAGCAGACATTGGAAG-39 (reverse primer), which contains an EcoRI site (underlined). RT-PCR was carried out with the following parameters: 94 °C denaturation for 4 min, 42 °C reverse transcription for 50 min, 94 °C denaturation for 2 min, and then 30 cycles of 94 °C denaturation for 30 s, 61 °C annealing for 30 s, 72 °C extension for 1 min, and final extension at 72 °C for 10 min. The amplified DNA fragment about 480 bp was detected by electrophoresis on 1.0% agarose gel.
Some colonies of the transformants were picked up ran domly from the plates and initially inoculated into a 50 ml conical tube containing 10 ml BMGY medium at 30 °C and 250 rpm. When the cultures reached OD600 = 2.0–6.0, the cells were harvested by centrifugation and resuspended by 10 ml BMMY medium to induce expression. The cells were allowed to grow for 72 h at 30 °C, and methanol was added every 24 h to a final concentration of 0.5% (v/v) for induced expression of the target protein. For recombinant protein detection, the culture filtrates of the transformants were run on a 15% (w/v) SDS–PAGE gel and stained with Coomassie blue R250. Optimized expression of rhbFGF in P. pastoris
Construction of expression vector pPICZa/hbFGF The resulting hbFGF cDNA fragment was digested with XhoI and EcoRI and then ligated to corresponding sites of the expression vector pPICZa. Then the ligation product was transformed into the competent cells of E. coli XL1Blue and the recombinant colonies were selected by scoring for zeocin (25 lg/ml) resistance. The procedures for small scale preparation of plasmid, digestion with restriction enzymes, ligation, and transformation all followed the stan dard methods.
In order to achieve high level yield of rhbFGF, diVerent culture parameters including pH value which was adjusted to pH 3.0–6.5 with 0.5 pH intervals, the optimal inducing time points and methanol daily addition concentration (0.25%, 0.5%, 1.0%, 1.5%) (v/v) were varied and evaluated in the expression procedure. The processes were the same as above said. At the desired time points, 0.2 ml cell aliquots were withdrawn and then replaced with equal amount of fresh medium. The supernatant sample was used to do ELISA assay.
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Enzyme-linked immunosorbent assay (ELISA)
Protein assay
Individual well of ELISA plate (Costar) was coated with 50 ll supernatant sample of rhbFGF and 50 ll coat ing buVer (15 mM Na2CO3, 35 mM NaHCO3, pH 9.6) over night at 4 °C. The plates were blocked with 5% (w/v) nonfat milk powder in TPBS (PBS with 0.05% Tween 20) and incubated in the coated plates for 2 h at room temperature. The rabbit anti-human bFGF polyclonal antibody (rabbit IgG, Boshide, China) was used at 1/1000, incubated 1 h at 37 °C. Following several washes with TPBS, the plates were incubated with goat anti-rabbit IgG conjugated to HRP (Dingguo, China) (1:1000 dilution at blocking buVer) for 1 h again. The color reaction was developed by addition of the substrate solution ortho-phenylenediamine (OPD) and incubated for 5 min at room temperature, and in the dark. Then 50 ll Stop Solution (2 mol/l H2SO4) was added to each well. The absorbance values at 490 nm were read in ELX800 Microplate Reader (Bio-Tek Instruments Inc.). The plate was read within 2 h after adding the Stop Solution.
The protein concentrations in the samples were deter mined with Bradford protein assay [14] using bovine serum albumin as the concentration standard.
Large-scale expression and purification of rhbFGF
Activity assay of rhbFGF
The best transformant clone was cultured in 2 L BMGY medium at 30 °C (pH 5.0) until the culture reached OD600 = 2.0–6.0, the cells were harvested by centrifugation and resuspended in 2 L BMMY medium, and cultivated at 30 °C with shaking for 3 days. The culture was supple mented daily with 10 ml methanol. A SP Sepharose column (20 ml, Pharmacia Biotech, Swe den) was equilibrated with 100 ml 20 mmol/l NaAc–HAc (pH 4.0) buVer. The supernatant was harvested by centrifu gation (3000g, 10 min, 4 °C) and was diluted with 200 mmol/ l NaAc–HAc (pH 4.0) to threefold, then was loaded onto the SP Sepharose column at the rate of 0.5 ml/min. The col umn was washed extensively with the same buVer at the rate of 1 ml/min. The bound protein was eluted with a lin ear salt gradient (0.5–1 M NaCl) while the flow rate was maintained at the rate of 1 ml/min. Aliquots were collected from the various fractions across the major peak. The elu tion fractions containing the rhbFGF were loaded onto a source™ 30RPC column (2.0 £ 15 cm, Pharmacia Biotech, Sweden) equilibrated with 0.1% TFA for the further purifi cation. The column was washed with a linear gradient of methanol (containing 0.1% TFA) from 10% to 100% over 2 h and the protein was monitored by measuring the UV absorbency at 214 nm. The pooled elution fractions contain ing rhbFGF from source™ 30RPC column were analyzed by SDS–PAGE. Gel densitometry was used to quantify the proportion of purified proteins among the eluates. Column eZuent containing rhbFGF was concentrated by vacuum distillation and methanol was almost completely removed. Finally, the purified protein was dissolved in PBS solution and stored at ¡20 °C for detection of bioactivity. The purified rhbFGF was carried out on a HPLC system (Waters 600E, USA) using a C18 reverse phase column for purity analysis.
To verify that rhbFGF produced and purified from P. pastoris had a stimulatory eVect on the proliferation of NIH/3T3 cells. The proliferation eVects of rhbFGF were determined by the MTT assay described by Xia [16]. MTT was dissolved in PBS at a concentration of 5 mg/ml and filtered. NIH/3T3 cells were seeded in flat-bottom, 96-well plates at an initial density of 5 £ 104 cells per ml (50 ll per well) and cultured in DMEM medium supplemented with 1.5% FCS and incubated for 12–24 h at 37 °C. Then, the purified rhbFGF of diVerent concentrations were added to the wells, and the final volumes were 100 ll. After 3 days incubation with rhbFGF, 20 ll MTT solution was added to each well. Then incubated for a further 4 h at 37 °C, the culture medium including MTT solution in the well was removed, and 150 ll DMSO was added to each well and mixed thoroughly to dissolve the crystals. The plates were read at 492 nm in a Microplate Reader model 450 (Bio-Rad Instruments, USA) to obtain the absorbance values. And cell proliferation was determined. The cells incubated with PBS were used as control. Specific activity was determined with reference to standard hbFGF (internal standard of 4000 IU/ml, from national institute for the control of phar maceutical and biological products, China). The experiment was repeated six times.
SDS–PAGE and Western blotting assays SDS–PAGE analysis was performed using a 15% gel according to the method of Laemmli [15]. For Western blot ting, proteins in the gel were transferred to a polyvinylidene difluoride membrane using a semi-dry electroblotting appa ratus (Bio-Rad) at 15 V for 30 min in 25 mM Tris–192 mM glycine. The membrane was blocked by incubating with solu tion containing 5% BSA for 1 h, and then incubated with the rabbit anti-human bFGF polyclonal antibody (Boshide, China). After being washed, the membrane was incubated with the goat anti-rabbit IgG conjugated to HRP (Dingguo, China), diluted 1:250. The bound antibody was detected using 3,39-diaminobenzidine (DAB).
Results and discussion Construction and transformation of pPICZa/hbFGF To obtain the hbFGF gene, RT-PCR was used to get the cDNA of hbFGF from human glioma cells. A specific DNA fragment about 480 bp was produced. The sequencing result (data not shown) of the PCR product confirmed that there was no diVerence from the previously documented
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sequence which was shown on NCBI (GenBank Accession No. NM002006). The cDNA fragment encoding the gene hbFGF was inserted between the XhoI and EcoRI sites of P. pastoris expression vector pPICZa. After transformation, the transformants were screened through the restriction enzymes HindIII and XbaI and were sequenced. The results of restriction analysis and DNA sequencing (data not shown) showed that the hbFGF gene was inserted correctly into the expression vector. Transformation with SacI-linearized ver sion of pPICZa/hbFGF, favored its insertion into the yeast genome by homologous recombination. Ninety percent of transformants were Mut+. However, with the presence of the AOX1 sequence in the plasmid, there was a chance that recombinant could occur at the AOX1 locus to disrupt the wild-type AOX1 gene and create Muts transformants. The use of genomic PCR analysis ensured the isolation of pure clones of transformants bearing the genomically inte grated copies of pPICZa/hbFGF plasmids. The PCR results showed there was an insert fragment of 500 bp for positive yeast transformants, while no insert fragment for negative yeast transformants (data not shown). Expression and detection of rhbFGF in P. pastoris In this study, the full-length hbFGF was inserted into the downstream of AOX1 promoter of the secretory expres sion vector pPICZaA, which carried a yeast a-factor signal sequence, and the engineering yeast expressing rhbFGF was successfully acquired. The secretory signal sequence a-factor
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and hbFGF were expressed in fusion forms and the fusion pro tein was cleaved at kex-2 cleavage site downstream of the signal sequence which targeting the fusion protein to the medium. The clone of the best activity was chosen for up scaled protein production. By analysis of the pre-expression and optimized expression process, the optimal culturing condi tions were achieved as follows: the pH optimum of 5.0 (Fig. 1a), the optimal induction time points at about the 3rd day for the strain (Fig. 1b) and methanol daily addition con centration of 0.5% (v/v) (Fig. 1c). Under these conditions, high level expression transformant of P. pastoris strain was obtained and the transformant with the highest yield of rhbFGF was retained for further studies. The culture supernatant of high expression transformant which was induced for 3 days was analyzed by SDS–PAGE. Based on the amino acid sequence, the calculated molecular weight of rhbFGF is 17.2 kDa, consistent with the result of SDS–PAGE measurement (Fig. 2a). The expression level of rhbFGF was estimated to be about 150 mg/l culture. Due to secretion of proteases to the medium, and possibly also due to released by lysis, proteolytic degradation is a significant problem in many high cell-density cultures. In P. pastoris, incubation temperatures of 30 °C, 25 °C, and 20 °C have been examined in attempt to minimize extracellular prote olysis. Low temperature reduced protease levels and greatly enhanced the yield of biologically active protein in P. pasto ris [17,18]. We found that decreasing the temperature from 30 °C to 20 °C during the methanol feed phase increased the yield of the recombinant protein about 1.5-fold.
Fig. 1. Expression optimization of X33 transformants for induction of rhbFGF. Supernatants collected at each evaluated condition were processed by ELISA. (a) Optimization of the pH value. (b) Optimization of the methanol induction time points. (c) Optimization of the methanol daily addition concen tration by measuring OD600.
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Fig. 2. (a) SDS–PAGE analysis of purified rhbFGF. Lane 1, protein molecular weight marker (low); lane 2, fractions passed through SP Sepharose gel; lane 3, fractions passed through source™ 30RPC. (b) Western blotting analysis of expressed protein in P. pastoris. Lanes 1 and 2, rhbFGF from diVerent protein concentrations; lane 3, standard hbFGF as a positive control; lane 4,
The characterization of purified rhbFGF The protein was purified from the supernatant by SP Sepharose ion exchange and source™ 30RPC chromatogra phy (Fig. 2a). The purity of rhbFGF was more than 94% in RP-HPLC. Bradford protein assay showed about 300 mg rhbFGF was obtained from 2 L cultivation broth, the final recovery of the recombinant protein was 61.5% (Table 1). The primary purified recombinant protein was identified by Western blot analysis. The results demonstrated that the recombinant protein could bind with the rabbit anti-human bFGF polyclonal antibody. No band was observed in lane 4, which was the supernatant of pPICZa transformant of X33 (Fig. 2b). The biological activity of rhbFGF was detected by the MTT method. The results demonstrated that the recombinant protein could stimulate the proliferation of NIH/3T3 cells very obviously. rhbFGF concentrations from 0.01 to 1000 ng/ml all showed a dose-dependent pro liferative eVect on NIH/3T3 cells (Fig. 3). This suggested that rhbFGF expressed by P. pastoris had a similar biologic function to standard hbFGF (national institute for the con trol of pharmaceutical and biological products, China). The specific activity of the biologically active protein was
Fig. 3. Stimulatory activities of rhbFGF on the proliferation of NIH/3T3 cells compared to a positive control. Grey bars, purified rhbFGF from the supernatant; black bars, standard hbFGF preparations. The mitogenic activity was estimated as the number of cells per well after incubation with each sample: rhbFGF or the positive control (standard hbFGF prepara tions). The experiment was repeated six times for each sample, the results revealed that the diVerence was significant (P < 0.05).
found to be 4.3 £ 106 IU/mg, which was almost commen surate with typical values (4.7 £ 106 IU/mg) obtained with standard hbFGF preparations demonstrating that correct folding had taken place. Purified rhbFGF showed no signif icant loss in its activity when stored for more than 4 weeks at ¡20 °C at pH 7.0.
Table 1 Summary of purification steps of rhbFGF from P. pastoris Purification steps Supernatants SP Sepharose XL Source™ 30RPC
Volume (L) 2 0.4 0.14
Total protein (mg/l) 276 203 150
Specific activity (U/mg) 6
4.1 £ 10 4.3 £ 106 4.3 £ 106
Total activity (U) 9
2.28 £ 10 1.77 £ 109 1.31 £ 109
Recovery (%) 100 77.6 61.5
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Discussion Of the current 22 homologues of the FGF polypeptide family, FGF-2 or basic fibroblast growth factor (bFGF) has a relatively high potential for clinical and pharmaceutical applications because of its specificity for epithelial cells medi ated through specific FGFR isoform FGFR2IIIb [19,20], its unique heparin-binding domain [21], and its apparent spec ificity for the antithrombin-binding, anticoagulant motif within heparin sulfate [22]. These potential applications require, at reasonable cost, large-scale quantities of purified recombinant polypeptide, both for its application directly for cytokine activity on epithelial cells and also for its indi rect use as an aYnity reagent for production and neutraliza tion of specific heparin sulfate motifs. Several hosts such as insect cell [23], E. coli [10] and Sac charomyces cerevisiae [24] were used for the expressions of rhbFGF. But the popular practice was that insertion of a short exogenous gene in upstream of hbFGF gene to produce rhbFGF. Used this fusion technique, the host rhbFGF peptide could be stabilized and expressed eVec tively. However, fusion rhbFGF was limited to only exter nal use in clinic due to its potential immunoreaction. There fore, nonfusion rhbFGF was preferred. Unfortunately, no nonfusion rhbFGF strain with expression rate of above 10% of total cellular protein was available [23]. And the E. coli expressed rhbFGF was present initially in inclusion bodies and bioactivity was realized only after renaturation, which complicated the purification process and led to low yield of product [23]. Furthermore, clinical application of bacterially produced products may be aVected by the potential presence of endotoxins that sometimes contami nate E. coli-expressed protein preparations [25]. Although S. cerevisiae has yielded pure, yields were not reported [24]. We have instead employed the methyloptrophic yeast P. pastoris, which have been shown to be a more eVective pro duction host than S. cerevisiae [26]. Compared with the above three expression systems, the P. pastoris system not only has the features of eukaryotic protein synthesis and modification pathway, but also has its own characteristic advantages: extremely high level expression of intra- or extracellular proteins; ease of genetic manipulation; ease of fermentation to high cell density and ease of the down stream purification on account of the fewer endogenous proteins secreted [11,27]. These properties have already made it the easiest and most eVective expression system with the potential to achieve full activity of the desired protein and suYciently display its great importance in the industrial, clinical, and scientific research fields [28,29]. The high oxygen demand of P. pastoris is best satisfied when grown in a fermentor, where parameters such as pH and carbon source feed can also be controlled. As a conse quence, optimal growth and induction are achieved under these conditions. Since the production of recombinant pro teins in P. pastoris is growth-related, it is of utmost impor tance to optimize biomass production to obtain high levels of the protein of interest [30]. To optimize the production
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of rhbFGF in P. pastoris, the strain could be cultured in a fermentor where the pH, aeration, agitation, and the metha nol feed are all tightly controlled. This will result in a nota bly improved total production level of the recombinant pro tein which to a larger extent is explained by the higher cell density achieved by fermentor growth. If taking this into account, the specific yield of rhbFGF would be much higher than 150 mg/l in a fermentor. In conclusion, we have developed an eYcient and func tional expression system (methylotrophic yeast P. pastoris) for rhbFGF in this report, and this system may not only facilitate further studies of rhbFGF in the near future, but also can allow possible large-scale production of biologi cally active rhbFGF . Acknowledgments We thank Dr. Weiqun Yan for critical reading of the manuscript. This work was supported by Grants from the National High Technology Research and Development Pro gram (863 program) of China (No. 2004AA205020). References [1] D.M. Ornitz, N. Itoh, Fibroblast growth factors, Genome Biol. 23 (2001) 1–12. [2] C. Basilico, D. Moscatelli, The FGF family of growth factors and onco genes, Adv. Cancer Res. 59 (1992) 115–165. [3] S.M. Schwartz, L. Liaw, Growth control and morphogenesis in the development and pathology of arteries, J. Cardiovasc. Pharmacol. 121 (Suppl.) (1993) S31–S49. [4] A. Bikfalvi, Z.C. Han, Angiogenic growth factors are hematopoietic growth factors and vice versa, Leukemia 8 (1994) 523–529. [5] A. Logan, A.S. Frautschy, A. Baird, Basic fibroblast growth factor and central nervous system injury, Ann. NY Acad. Sci. USA 63 (1991) 474–476. [6] K. Unsicker, S. Engels, C. Hamm, G. Ludecke, C. Meier, J. Renzing, H.G. Terbrack, K. Flanders, Molecular control of neural plasticity by the multifunctional growth factor families of the FGFs and TGF-b, Anat. Anz. 174 (1992) 405–407. [7] J.M. McAvoy, G.C. Chamberlain, R.V. de Longh, N.A. Richardson, F.J. Lovicu, The role of fibroblast growth factor in eye lensdevelop ment, Ann. NY Acad. Sci. 638 (1991) 256–274. [8] B.B. Riley, M.P. Savage, B.K. Simandl, B.B. Olwin, J.F. Fallon, Retrovi ral expression of FGF-2 (bFGF) aVects patterning in chick limb bud, Development 118 (1993) 95–104. [9] X. Liu, X.Z. Zhu, Basic fibroblast growth factor protected forebrain against ischemia-reperfusion damage in rats, Acta Pharmacol. Sin. 19 (1996) 527–530. [10] K. Youqiang, C. WilkinsonM, D.G. Feming, A rapid procedure for production of human basic fibroblast growth factor in Escherichia coli cells, Biochim. Biophys. Acta 1131 (1992) 307–310. [11] S.M. Patrick, M.L. Fazenda, B.M. Neil, L.M. Harvey, Heterologous protein production using the Pichia pastoris expression system, Yeast 22 (2005) 249–270. [12] C. Gurkan, D.J. Ellar, Recombinant production of bacterial toxins and their derivatives in the methylotrophic yeast Pichia pastoris, Mic rob. Cell Fact. 4 (2005) 2033. [13] Invitrogen, A manual of methods for expression of recombinant pro teins in Pichia pastoris, Catalog No. K1710-01. [14] M.M. Bradford, A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein–dye binding, Anal. Biochem. 72 (1976) 248–254.
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[15] U.K. Laemmli, Cleavage of structural proteins during assembly of the head of bacteriophage T4, Nature 277 (1970) 680–685. [16] L. Xia, EVects of epidermal growth factor on the growth of human gastric cancer cell and the implanted tumor of nude mice, World J. Gastroenterol. 8 (2002) 455–458. [17] X. Shi, T. Karkut, M. Chamankhah, M. Alting-Mees, S.M. Hemming sen, D. Hegedus, Optimal conditions for the expression of a singlechain antibody (scFv) gene in Pichia pastoris, Protein Expr. Purif. 28 (2003) 321–330. [18] S.Z. Li, F. Xiong, Q. Lin, Low-temperature increases the yield of bio logically active herring antifreeze protein in Pichia pastoris, Protein Expr. Purif. 21 (2001) 438–445. [19] W.L. McKeehan, F. Wang, M. Kan, The heparan sulfate fibroblast growth factor family: diversity of structure and function, Prog. Nucleic Acid Res. Mol. Biol. 59 (1998) 135–176. [20] G. Yan, Y. Fukabori, G. McBride, S. Nikolaropolous, W.L. McKee han, Exon switching and activation of stromal and embryonic fibro blast growth factor (FGF)–FGF receptor genesin prostate epithelial cells accompany stromal independence and malignancy, Mol. Cell. Biol. 13 (1993) 4513–4522. [21] S. Ye, Y. Luo, W. Lu, R.B. Jones, R.J. Lindhardt, I. Capila, T. Toida, M. Kan, H. Pelletier, W.L. McKeehan, Structural basis for interaction of FGF-1, FGF-2, and FGF-7 with diVerent heparan sulfate motifs, Biochemistry 40 (2001) 14429–14439.
[22] Y. Luo, H.H. Cho, W.L. McKeehan, Biospecific extraction and neutral ization of anticoagulant heparin with fibroblast growth factors (FGF), J. Pharm. Sci. 92 (2003) 2117–2127. [23] Y.M. Wang, L.C. Sun, Y.H. Qi, Y. Si, Expression of human basic fibro blast growth factor gene in sf9 insect cell, J. Cap. Univ. Med. Sci. 25 (2004) 431–434. [24] J. Philip, S. Lawrence, B. Andrence, Expression and processing of bio logically active fibroblast growth factor in the yeast Saccharomyces cerevisiae, J. Biol. Chem. 263 (1988) 16471–16476. [25] L. Yan, Y.L. Qin, W.C. Bei, X.L. Yu, H.L. Huang, H.C. Chen, Expres sion of an interleukin-6-interleukin-2 fusion protein (pIL-6–IL-2) in P. pastoris, Eur. Cytokine Netw. 15 (2004) 240–246. [26] D.R. Higgins, J.M. Cregg, Introduction to Pichia pastoris, Methods Mol. Biol. 103 (1998) 1–15. [27] J.M. Cregg, T. Svedvick, W.C. Raschke, Recent advances in the expres sion of foreign genes in Pichia pastoris, Biotechniques 11 (1993) 905–910. [28] J.L. Cereghino, J.M. Cregg, Heterologous protein expression in the methylotrophic yeast Pichia pastoris, FEMS Microbiol. Rev. 24 (2000) 45–66. [29] M. Romanos, Advances in the use of Pichia pastoris for high-level gene expression, Biotechniques 6 (1995) 527–533. [30] G.P. Cereghino, Production of recombinant proteins in fermentor cul tures of the yeast Pichia pastoris, Curr. Opin. Biotechnol. 13 (4) (2002) 329–332.
Protein Expression and Purification 59 (2008) 282–288 www.elsevier.com/locate/yprep
High-level expression, purification, and characterization of recombinant human basic fibroblast growth factor in Pichia pastoris Xupeng Mu, Ning Kong, Weili Chen, Ting Zhang, Mohan Shen, Weiqun Yan * Department of Biochemistry, Institute of Frontier Medical Sciences, Jilin University, Xinmindajie Street 1163, Changchun, Jilin 130021, China Received 11 November 2007, and in revised form 14 Feburary 2008 Available online 29 February 2008
Abstract Basic fibroblast growth factor [basic FGF (bFGF); FGF-2] is an important member of the FGF family, bFGF is a potent angio genic molecule in vivo and in vitro stimulate smooth muscle cell growth, wound healing, and tissue repair. The full-length hbFGF coding sequence, gained by RT-PCR, was cloned into the pPICZaA vector in frame with the yeast a-factor secretion signal under the tran scriptional control of the AOX promoter and integrated into Pichia pastoris strain X33, and the high level expression of rhbFGF has been achieved. SDS–PAGE and Western blotting assays of culture broth from a methanol-induced expression strain demonstrated that rhbFGF, an 18 kDa protein, was secreted into the culture medium. The growth conditions of the transformant strain were optimized in 50 ml conical tubes including methanol concentration, pH and inducing time. Under the optimal conditions, stable production of rhbFGF around 150 mg/l was achieved. The expressed rhbFGF was purified more than 94% purity using SP Sepharose ion exchange chromatogra phy and source™ 30RPC. A preliminary biochemical characterization of purified rhbFGF was performed by biological activity analysis which was used by NIH/3T3 cell cultures, and the results demonstrated that the purified rhbFGF stimulated the growth of NIH/3T3 cells similarly to standard material. © 2008 Elsevier Inc. All rights reserved. Keywords: Basic fibroblast growth factor; Pichia pastoris; Secretory expression; Protein purification
The fibroblast growth factor (FGF)1 protein family of heparin-binding proteins currently consists of 22 structur ally-related polypeptides with similar biological activities. These include the two prototypes, FGF1 (aFGF) and FGF2 (bFGF) [1]. As a single-chain protein, bFGF has 146 amino acids and pI of 9.6, and Mr is about 17,200. Natural bFGF was isolated initially from pituitary extracts. BFGF has pleio tropic eVects in diVerent cells and organ systems. BFGF can stimulate smooth muscle cell growth, wound healing, and tissue repair [2,3]. In addition, bFGF may stimulate hemato poiesis [4] and may play an important role in the diVerentia tion and/or function of the nervous system [5,6], the eye [7], * Corresponding author. E-mail address: [email protected] (W. Yan). 1 Abbreviations used: bFGF, basic fibroblast growth factor; PBS, phos phate-buVered saline; OPD, ortho-phenylenediamine; DAB, 3,39-diam inobenzidine; FCS, fetal calf serum; BSA, bovine serum albumin; DMEM, Dulbecco’s modified Eagle’s medium; MTT, methylthiazoletetrazolium; DMSO, dimethyl sulfoxide. 1046-5928/$ - see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.pep.2008.02.009
and the skeleton [8]. In animal study, bFGF can prevent the brain from injury of ischemia and reperfusion in rats [9]. Therefore, obtaining large quantities of recombinant bFGF is important for both clinical studies and mechanistic investigations. But it was almost impossible to obtain suY cient bFGF from animal tissues due to its extremely low quantity and high expense. Therefore, in the last decade, genetic technology has been employed to produce bFGF at low cost. So far, recombinant bFGF which was used in clinic has been produced by Escherichia coli [10], the expres sion of rhbFGF by Pichia pastoris has not been reported yet. P. pastoris is a widely used, eYcient expression system for a wide variety of molecules [11]. P. pastoris is physically robust and amenable to high-density fermentation as E. coli but possess the necessary cellular machinery to carry out post-translational modifications. With the advantages of both prokaryotic and eukaryotic systems, P. pastoris provides the potential for producing soluble, correctly folded recombinant proteins that have
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undergone all the post-translational modifications required for functionality and is easy to handle and its fermentation conditions are very simple. Additionally, isolation of foreign protein is easy, because P. pastoris does not secrete a large amount of intrinsic protein [12]. In the present study, we have cloned the cDNA encoding hbFGF and achieved its high level secreting expression with a yield of 150 mg/l of yeast culture. Activity assay showed that rhbFGF had the biological function as the standard material. Materials and methods Cell, vector, host strains, and reagents Plasmids were amplified in E. coli XL1-Blue (Dingguo, China). P. pastoris and the P. pastoris integrative expression vector (pPICZaA) were obtained from Invitrogen (USA) and pPICZaA vector was reconstructed by our lab which did not have the two Ste13 cleavage sites. All media for growth of P. pastoris were prepared as the protocols obtained from man ufacturer (Invitrogen, USA). Restriction enzymes and T4 DNA ligase were purchased from Takara (Dalian, China). PCR purification kit, gel extraction kit, and Miniprep kit for plasmid extraction were obtained from Qiangen Company (USA). Primer was synthesized by Shenggong Company (Shanghai, China). Standard hbFGF was purchased from national institute for the control of pharmaceutical and bio logical products (China).
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Electroporation of X33 and screening for recombinant strain Plasmid DNA was linearized with SacI and was purified by phenol–chloroform extraction and ethanol precipitation. The purified DNA fragment, about 10 lg, was dissolved in 10 ll of ddH2O. Electrocompetent cells of P. pastoris X33 were prepared according to the supplier’s instruction [13]. Then 80 ll of competent cells were mixed with 10 lg line arized recombinant plasmid in a 0.2 cm electroporation cuvette. The mixtures were incubated on ice for 5 min, and the electroporation was carried out on GenePulser appara tus (Bio-Rad, USA) with the following settings: 1.5 kV volt age, 25 lF capacitance, and 200 X resistance. After pulsing, 1.0 ml 1 M ice-cold sorbitol was added immediately to the cuvette. The cells were transferred into a 1.5 ml sterile tube and incubated at 30 °C without shaking for 1 h. Then the transformed cells were plated on YPDS containing zeocin for 100 lg/ml and incubated at 30 °C for at least 3 days. Genomic DNA analysis To verify the recombinant gene integration, the g enomic DNA of a number of transformant recombinant P. pastoris strains were detected by PCR assay. The following primers were used: AOX1 universal primers (sense: 59-G ACTGGTTCCAATTGACAAGC-39, anti-sense: 59-GC AAATGGCATTCTGACATCC-39). Thirty cycles of ampli fication were carried out at 94 °C for 30 s, 55 °C for 30 s, and 72 °C for 1 min.
RNA extraction and RT-PCR Induced expression of rhbFGF in P. pastoris Total RNA was extracted from human glioma cells and used as the template for RT-PCR. The primers were 59-ATACTCGAGAAGAGAGCAGCCGGGAGCATCA CCA-39 (forward primer), which contains an XhoI site (underlined) and the kex2 site (underlined), and 59-GCGGA ATTCTCAGCTCTTAGCAGACATTGGAAG-39 (reverse primer), which contains an EcoRI site (underlined). RT-PCR was carried out with the following parameters: 94 °C denaturation for 4 min, 42 °C reverse transcription for 50 min, 94 °C denaturation for 2 min, and then 30 cycles of 94 °C denaturation for 30 s, 61 °C annealing for 30 s, 72 °C extension for 1 min, and final extension at 72 °C for 10 min. The amplified DNA fragment about 480 bp was detected by electrophoresis on 1.0% agarose gel.
Some colonies of the transformants were picked up ran domly from the plates and initially inoculated into a 50 ml conical tube containing 10 ml BMGY medium at 30 °C and 250 rpm. When the cultures reached OD600 = 2.0–6.0, the cells were harvested by centrifugation and resuspended by 10 ml BMMY medium to induce expression. The cells were allowed to grow for 72 h at 30 °C, and methanol was added every 24 h to a final concentration of 0.5% (v/v) for induced expression of the target protein. For recombinant protein detection, the culture filtrates of the transformants were run on a 15% (w/v) SDS–PAGE gel and stained with Coomassie blue R250. Optimized expression of rhbFGF in P. pastoris
Construction of expression vector pPICZa/hbFGF The resulting hbFGF cDNA fragment was digested with XhoI and EcoRI and then ligated to corresponding sites of the expression vector pPICZa. Then the ligation product was transformed into the competent cells of E. coli XL1Blue and the recombinant colonies were selected by scoring for zeocin (25 lg/ml) resistance. The procedures for small scale preparation of plasmid, digestion with restriction enzymes, ligation, and transformation all followed the stan dard methods.
In order to achieve high level yield of rhbFGF, diVerent culture parameters including pH value which was adjusted to pH 3.0–6.5 with 0.5 pH intervals, the optimal inducing time points and methanol daily addition concentration (0.25%, 0.5%, 1.0%, 1.5%) (v/v) were varied and evaluated in the expression procedure. The processes were the same as above said. At the desired time points, 0.2 ml cell aliquots were withdrawn and then replaced with equal amount of fresh medium. The supernatant sample was used to do ELISA assay.
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Enzyme-linked immunosorbent assay (ELISA)
Protein assay
Individual well of ELISA plate (Costar) was coated with 50 ll supernatant sample of rhbFGF and 50 ll coat ing buVer (15 mM Na2CO3, 35 mM NaHCO3, pH 9.6) over night at 4 °C. The plates were blocked with 5% (w/v) nonfat milk powder in TPBS (PBS with 0.05% Tween 20) and incubated in the coated plates for 2 h at room temperature. The rabbit anti-human bFGF polyclonal antibody (rabbit IgG, Boshide, China) was used at 1/1000, incubated 1 h at 37 °C. Following several washes with TPBS, the plates were incubated with goat anti-rabbit IgG conjugated to HRP (Dingguo, China) (1:1000 dilution at blocking buVer) for 1 h again. The color reaction was developed by addition of the substrate solution ortho-phenylenediamine (OPD) and incubated for 5 min at room temperature, and in the dark. Then 50 ll Stop Solution (2 mol/l H2SO4) was added to each well. The absorbance values at 490 nm were read in ELX800 Microplate Reader (Bio-Tek Instruments Inc.). The plate was read within 2 h after adding the Stop Solution.
The protein concentrations in the samples were deter mined with Bradford protein assay [14] using bovine serum albumin as the concentration standard.
Large-scale expression and purification of rhbFGF
Activity assay of rhbFGF
The best transformant clone was cultured in 2 L BMGY medium at 30 °C (pH 5.0) until the culture reached OD600 = 2.0–6.0, the cells were harvested by centrifugation and resuspended in 2 L BMMY medium, and cultivated at 30 °C with shaking for 3 days. The culture was supple mented daily with 10 ml methanol. A SP Sepharose column (20 ml, Pharmacia Biotech, Swe den) was equilibrated with 100 ml 20 mmol/l NaAc–HAc (pH 4.0) buVer. The supernatant was harvested by centrifu gation (3000g, 10 min, 4 °C) and was diluted with 200 mmol/ l NaAc–HAc (pH 4.0) to threefold, then was loaded onto the SP Sepharose column at the rate of 0.5 ml/min. The col umn was washed extensively with the same buVer at the rate of 1 ml/min. The bound protein was eluted with a lin ear salt gradient (0.5–1 M NaCl) while the flow rate was maintained at the rate of 1 ml/min. Aliquots were collected from the various fractions across the major peak. The elu tion fractions containing the rhbFGF were loaded onto a source™ 30RPC column (2.0 £ 15 cm, Pharmacia Biotech, Sweden) equilibrated with 0.1% TFA for the further purifi cation. The column was washed with a linear gradient of methanol (containing 0.1% TFA) from 10% to 100% over 2 h and the protein was monitored by measuring the UV absorbency at 214 nm. The pooled elution fractions contain ing rhbFGF from source™ 30RPC column were analyzed by SDS–PAGE. Gel densitometry was used to quantify the proportion of purified proteins among the eluates. Column eZuent containing rhbFGF was concentrated by vacuum distillation and methanol was almost completely removed. Finally, the purified protein was dissolved in PBS solution and stored at ¡20 °C for detection of bioactivity. The purified rhbFGF was carried out on a HPLC system (Waters 600E, USA) using a C18 reverse phase column for purity analysis.
To verify that rhbFGF produced and purified from P. pastoris had a stimulatory eVect on the proliferation of NIH/3T3 cells. The proliferation eVects of rhbFGF were determined by the MTT assay described by Xia [16]. MTT was dissolved in PBS at a concentration of 5 mg/ml and filtered. NIH/3T3 cells were seeded in flat-bottom, 96-well plates at an initial density of 5 £ 104 cells per ml (50 ll per well) and cultured in DMEM medium supplemented with 1.5% FCS and incubated for 12–24 h at 37 °C. Then, the purified rhbFGF of diVerent concentrations were added to the wells, and the final volumes were 100 ll. After 3 days incubation with rhbFGF, 20 ll MTT solution was added to each well. Then incubated for a further 4 h at 37 °C, the culture medium including MTT solution in the well was removed, and 150 ll DMSO was added to each well and mixed thoroughly to dissolve the crystals. The plates were read at 492 nm in a Microplate Reader model 450 (Bio-Rad Instruments, USA) to obtain the absorbance values. And cell proliferation was determined. The cells incubated with PBS were used as control. Specific activity was determined with reference to standard hbFGF (internal standard of 4000 IU/ml, from national institute for the control of phar maceutical and biological products, China). The experiment was repeated six times.
SDS–PAGE and Western blotting assays SDS–PAGE analysis was performed using a 15% gel according to the method of Laemmli [15]. For Western blot ting, proteins in the gel were transferred to a polyvinylidene difluoride membrane using a semi-dry electroblotting appa ratus (Bio-Rad) at 15 V for 30 min in 25 mM Tris–192 mM glycine. The membrane was blocked by incubating with solu tion containing 5% BSA for 1 h, and then incubated with the rabbit anti-human bFGF polyclonal antibody (Boshide, China). After being washed, the membrane was incubated with the goat anti-rabbit IgG conjugated to HRP (Dingguo, China), diluted 1:250. The bound antibody was detected using 3,39-diaminobenzidine (DAB).
Results and discussion Construction and transformation of pPICZa/hbFGF To obtain the hbFGF gene, RT-PCR was used to get the cDNA of hbFGF from human glioma cells. A specific DNA fragment about 480 bp was produced. The sequencing result (data not shown) of the PCR product confirmed that there was no diVerence from the previously documented
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sequence which was shown on NCBI (GenBank Accession No. NM002006). The cDNA fragment encoding the gene hbFGF was inserted between the XhoI and EcoRI sites of P. pastoris expression vector pPICZa. After transformation, the transformants were screened through the restriction enzymes HindIII and XbaI and were sequenced. The results of restriction analysis and DNA sequencing (data not shown) showed that the hbFGF gene was inserted correctly into the expression vector. Transformation with SacI-linearized ver sion of pPICZa/hbFGF, favored its insertion into the yeast genome by homologous recombination. Ninety percent of transformants were Mut+. However, with the presence of the AOX1 sequence in the plasmid, there was a chance that recombinant could occur at the AOX1 locus to disrupt the wild-type AOX1 gene and create Muts transformants. The use of genomic PCR analysis ensured the isolation of pure clones of transformants bearing the genomically inte grated copies of pPICZa/hbFGF plasmids. The PCR results showed there was an insert fragment of 500 bp for positive yeast transformants, while no insert fragment for negative yeast transformants (data not shown). Expression and detection of rhbFGF in P. pastoris In this study, the full-length hbFGF was inserted into the downstream of AOX1 promoter of the secretory expres sion vector pPICZaA, which carried a yeast a-factor signal sequence, and the engineering yeast expressing rhbFGF was successfully acquired. The secretory signal sequence a-factor
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and hbFGF were expressed in fusion forms and the fusion pro tein was cleaved at kex-2 cleavage site downstream of the signal sequence which targeting the fusion protein to the medium. The clone of the best activity was chosen for up scaled protein production. By analysis of the pre-expression and optimized expression process, the optimal culturing condi tions were achieved as follows: the pH optimum of 5.0 (Fig. 1a), the optimal induction time points at about the 3rd day for the strain (Fig. 1b) and methanol daily addition con centration of 0.5% (v/v) (Fig. 1c). Under these conditions, high level expression transformant of P. pastoris strain was obtained and the transformant with the highest yield of rhbFGF was retained for further studies. The culture supernatant of high expression transformant which was induced for 3 days was analyzed by SDS–PAGE. Based on the amino acid sequence, the calculated molecular weight of rhbFGF is 17.2 kDa, consistent with the result of SDS–PAGE measurement (Fig. 2a). The expression level of rhbFGF was estimated to be about 150 mg/l culture. Due to secretion of proteases to the medium, and possibly also due to released by lysis, proteolytic degradation is a significant problem in many high cell-density cultures. In P. pastoris, incubation temperatures of 30 °C, 25 °C, and 20 °C have been examined in attempt to minimize extracellular prote olysis. Low temperature reduced protease levels and greatly enhanced the yield of biologically active protein in P. pasto ris [17,18]. We found that decreasing the temperature from 30 °C to 20 °C during the methanol feed phase increased the yield of the recombinant protein about 1.5-fold.
Fig. 1. Expression optimization of X33 transformants for induction of rhbFGF. Supernatants collected at each evaluated condition were processed by ELISA. (a) Optimization of the pH value. (b) Optimization of the methanol induction time points. (c) Optimization of the methanol daily addition concen tration by measuring OD600.
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Fig. 2. (a) SDS–PAGE analysis of purified rhbFGF. Lane 1, protein molecular weight marker (low); lane 2, fractions passed through SP Sepharose gel; lane 3, fractions passed through source™ 30RPC. (b) Western blotting analysis of expressed protein in P. pastoris. Lanes 1 and 2, rhbFGF from diVerent protein concentrations; lane 3, standard hbFGF as a positive control; lane 4,
The characterization of purified rhbFGF The protein was purified from the supernatant by SP Sepharose ion exchange and source™ 30RPC chromatogra phy (Fig. 2a). The purity of rhbFGF was more than 94% in RP-HPLC. Bradford protein assay showed about 300 mg rhbFGF was obtained from 2 L cultivation broth, the final recovery of the recombinant protein was 61.5% (Table 1). The primary purified recombinant protein was identified by Western blot analysis. The results demonstrated that the recombinant protein could bind with the rabbit anti-human bFGF polyclonal antibody. No band was observed in lane 4, which was the supernatant of pPICZa transformant of X33 (Fig. 2b). The biological activity of rhbFGF was detected by the MTT method. The results demonstrated that the recombinant protein could stimulate the proliferation of NIH/3T3 cells very obviously. rhbFGF concentrations from 0.01 to 1000 ng/ml all showed a dose-dependent pro liferative eVect on NIH/3T3 cells (Fig. 3). This suggested that rhbFGF expressed by P. pastoris had a similar biologic function to standard hbFGF (national institute for the con trol of pharmaceutical and biological products, China). The specific activity of the biologically active protein was
Fig. 3. Stimulatory activities of rhbFGF on the proliferation of NIH/3T3 cells compared to a positive control. Grey bars, purified rhbFGF from the supernatant; black bars, standard hbFGF preparations. The mitogenic activity was estimated as the number of cells per well after incubation with each sample: rhbFGF or the positive control (standard hbFGF prepara tions). The experiment was repeated six times for each sample, the results revealed that the diVerence was significant (P < 0.05).
found to be 4.3 £ 106 IU/mg, which was almost commen surate with typical values (4.7 £ 106 IU/mg) obtained with standard hbFGF preparations demonstrating that correct folding had taken place. Purified rhbFGF showed no signif icant loss in its activity when stored for more than 4 weeks at ¡20 °C at pH 7.0.
Table 1 Summary of purification steps of rhbFGF from P. pastoris Purification steps Supernatants SP Sepharose XL Source™ 30RPC
Volume (L) 2 0.4 0.14
Total protein (mg/l) 276 203 150
Specific activity (U/mg) 6
4.1 £ 10 4.3 £ 106 4.3 £ 106
Total activity (U) 9
2.28 £ 10 1.77 £ 109 1.31 £ 109
Recovery (%) 100 77.6 61.5
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Discussion Of the current 22 homologues of the FGF polypeptide family, FGF-2 or basic fibroblast growth factor (bFGF) has a relatively high potential for clinical and pharmaceutical applications because of its specificity for epithelial cells medi ated through specific FGFR isoform FGFR2IIIb [19,20], its unique heparin-binding domain [21], and its apparent spec ificity for the antithrombin-binding, anticoagulant motif within heparin sulfate [22]. These potential applications require, at reasonable cost, large-scale quantities of purified recombinant polypeptide, both for its application directly for cytokine activity on epithelial cells and also for its indi rect use as an aYnity reagent for production and neutraliza tion of specific heparin sulfate motifs. Several hosts such as insect cell [23], E. coli [10] and Sac charomyces cerevisiae [24] were used for the expressions of rhbFGF. But the popular practice was that insertion of a short exogenous gene in upstream of hbFGF gene to produce rhbFGF. Used this fusion technique, the host rhbFGF peptide could be stabilized and expressed eVec tively. However, fusion rhbFGF was limited to only exter nal use in clinic due to its potential immunoreaction. There fore, nonfusion rhbFGF was preferred. Unfortunately, no nonfusion rhbFGF strain with expression rate of above 10% of total cellular protein was available [23]. And the E. coli expressed rhbFGF was present initially in inclusion bodies and bioactivity was realized only after renaturation, which complicated the purification process and led to low yield of product [23]. Furthermore, clinical application of bacterially produced products may be aVected by the potential presence of endotoxins that sometimes contami nate E. coli-expressed protein preparations [25]. Although S. cerevisiae has yielded pure, yields were not reported [24]. We have instead employed the methyloptrophic yeast P. pastoris, which have been shown to be a more eVective pro duction host than S. cerevisiae [26]. Compared with the above three expression systems, the P. pastoris system not only has the features of eukaryotic protein synthesis and modification pathway, but also has its own characteristic advantages: extremely high level expression of intra- or extracellular proteins; ease of genetic manipulation; ease of fermentation to high cell density and ease of the down stream purification on account of the fewer endogenous proteins secreted [11,27]. These properties have already made it the easiest and most eVective expression system with the potential to achieve full activity of the desired protein and suYciently display its great importance in the industrial, clinical, and scientific research fields [28,29]. The high oxygen demand of P. pastoris is best satisfied when grown in a fermentor, where parameters such as pH and carbon source feed can also be controlled. As a conse quence, optimal growth and induction are achieved under these conditions. Since the production of recombinant pro teins in P. pastoris is growth-related, it is of utmost impor tance to optimize biomass production to obtain high levels of the protein of interest [30]. To optimize the production
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of rhbFGF in P. pastoris, the strain could be cultured in a fermentor where the pH, aeration, agitation, and the metha nol feed are all tightly controlled. This will result in a nota bly improved total production level of the recombinant pro tein which to a larger extent is explained by the higher cell density achieved by fermentor growth. If taking this into account, the specific yield of rhbFGF would be much higher than 150 mg/l in a fermentor. In conclusion, we have developed an eYcient and func tional expression system (methylotrophic yeast P. pastoris) for rhbFGF in this report, and this system may not only facilitate further studies of rhbFGF in the near future, but also can allow possible large-scale production of biologi cally active rhbFGF . Acknowledgments We thank Dr. Weiqun Yan for critical reading of the manuscript. This work was supported by Grants from the National High Technology Research and Development Pro gram (863 program) of China (No. 2004AA205020). References [1] D.M. Ornitz, N. Itoh, Fibroblast growth factors, Genome Biol. 23 (2001) 1–12. [2] C. Basilico, D. Moscatelli, The FGF family of growth factors and onco genes, Adv. Cancer Res. 59 (1992) 115–165. [3] S.M. Schwartz, L. Liaw, Growth control and morphogenesis in the development and pathology of arteries, J. Cardiovasc. Pharmacol. 121 (Suppl.) (1993) S31–S49. [4] A. Bikfalvi, Z.C. Han, Angiogenic growth factors are hematopoietic growth factors and vice versa, Leukemia 8 (1994) 523–529. [5] A. Logan, A.S. Frautschy, A. Baird, Basic fibroblast growth factor and central nervous system injury, Ann. NY Acad. Sci. USA 63 (1991) 474–476. [6] K. Unsicker, S. Engels, C. Hamm, G. Ludecke, C. Meier, J. Renzing, H.G. Terbrack, K. Flanders, Molecular control of neural plasticity by the multifunctional growth factor families of the FGFs and TGF-b, Anat. Anz. 174 (1992) 405–407. [7] J.M. McAvoy, G.C. Chamberlain, R.V. de Longh, N.A. Richardson, F.J. Lovicu, The role of fibroblast growth factor in eye lensdevelop ment, Ann. NY Acad. Sci. 638 (1991) 256–274. [8] B.B. Riley, M.P. Savage, B.K. Simandl, B.B. Olwin, J.F. Fallon, Retrovi ral expression of FGF-2 (bFGF) aVects patterning in chick limb bud, Development 118 (1993) 95–104. [9] X. Liu, X.Z. Zhu, Basic fibroblast growth factor protected forebrain against ischemia-reperfusion damage in rats, Acta Pharmacol. Sin. 19 (1996) 527–530. [10] K. Youqiang, C. WilkinsonM, D.G. Feming, A rapid procedure for production of human basic fibroblast growth factor in Escherichia coli cells, Biochim. Biophys. Acta 1131 (1992) 307–310. [11] S.M. Patrick, M.L. Fazenda, B.M. Neil, L.M. Harvey, Heterologous protein production using the Pichia pastoris expression system, Yeast 22 (2005) 249–270. [12] C. Gurkan, D.J. Ellar, Recombinant production of bacterial toxins and their derivatives in the methylotrophic yeast Pichia pastoris, Mic rob. Cell Fact. 4 (2005) 2033. [13] Invitrogen, A manual of methods for expression of recombinant pro teins in Pichia pastoris, Catalog No. K1710-01. [14] M.M. Bradford, A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein–dye binding, Anal. Biochem. 72 (1976) 248–254.
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[15] U.K. Laemmli, Cleavage of structural proteins during assembly of the head of bacteriophage T4, Nature 277 (1970) 680–685. [16] L. Xia, EVects of epidermal growth factor on the growth of human gastric cancer cell and the implanted tumor of nude mice, World J. Gastroenterol. 8 (2002) 455–458. [17] X. Shi, T. Karkut, M. Chamankhah, M. Alting-Mees, S.M. Hemming sen, D. Hegedus, Optimal conditions for the expression of a singlechain antibody (scFv) gene in Pichia pastoris, Protein Expr. Purif. 28 (2003) 321–330. [18] S.Z. Li, F. Xiong, Q. Lin, Low-temperature increases the yield of bio logically active herring antifreeze protein in Pichia pastoris, Protein Expr. Purif. 21 (2001) 438–445. [19] W.L. McKeehan, F. Wang, M. Kan, The heparan sulfate fibroblast growth factor family: diversity of structure and function, Prog. Nucleic Acid Res. Mol. Biol. 59 (1998) 135–176. [20] G. Yan, Y. Fukabori, G. McBride, S. Nikolaropolous, W.L. McKee han, Exon switching and activation of stromal and embryonic fibro blast growth factor (FGF)–FGF receptor genesin prostate epithelial cells accompany stromal independence and malignancy, Mol. Cell. Biol. 13 (1993) 4513–4522. [21] S. Ye, Y. Luo, W. Lu, R.B. Jones, R.J. Lindhardt, I. Capila, T. Toida, M. Kan, H. Pelletier, W.L. McKeehan, Structural basis for interaction of FGF-1, FGF-2, and FGF-7 with diVerent heparan sulfate motifs, Biochemistry 40 (2001) 14429–14439.
[22] Y. Luo, H.H. Cho, W.L. McKeehan, Biospecific extraction and neutral ization of anticoagulant heparin with fibroblast growth factors (FGF), J. Pharm. Sci. 92 (2003) 2117–2127. [23] Y.M. Wang, L.C. Sun, Y.H. Qi, Y. Si, Expression of human basic fibro blast growth factor gene in sf9 insect cell, J. Cap. Univ. Med. Sci. 25 (2004) 431–434. [24] J. Philip, S. Lawrence, B. Andrence, Expression and processing of bio logically active fibroblast growth factor in the yeast Saccharomyces cerevisiae, J. Biol. Chem. 263 (1988) 16471–16476. [25] L. Yan, Y.L. Qin, W.C. Bei, X.L. Yu, H.L. Huang, H.C. Chen, Expres sion of an interleukin-6-interleukin-2 fusion protein (pIL-6–IL-2) in P. pastoris, Eur. Cytokine Netw. 15 (2004) 240–246. [26] D.R. Higgins, J.M. Cregg, Introduction to Pichia pastoris, Methods Mol. Biol. 103 (1998) 1–15. [27] J.M. Cregg, T. Svedvick, W.C. Raschke, Recent advances in the expres sion of foreign genes in Pichia pastoris, Biotechniques 11 (1993) 905–910. [28] J.L. Cereghino, J.M. Cregg, Heterologous protein expression in the methylotrophic yeast Pichia pastoris, FEMS Microbiol. Rev. 24 (2000) 45–66. [29] M. Romanos, Advances in the use of Pichia pastoris for high-level gene expression, Biotechniques 6 (1995) 527–533. [30] G.P. Cereghino, Production of recombinant proteins in fermentor cul tures of the yeast Pichia pastoris, Curr. Opin. Biotechnol. 13 (4) (2002) 329–332.