A pH-Induced, Intein-Mediated Expression and Purification of Recombinant Human Epidermal Growth Factor in Escherichia coli Yuejuan Zhang, Kun Zhang, Yi Wan, Jing Zi, Yan Wang, and Jun Wang Inst. of Microbiology, Shaanxi Province Academy of Sciences, Xi’an 710043, China
Lili Wang Key Laboratory of Synthetic and Natural Functional Molecule Chemistry of Ministry of Education, Shaanxi Key Laboratory of Modern Separation Science, Institute of Modern Separation Science, Northwest University, Xi’an 710069, China
Xiaochang Xue State Key Laboratory of Cancer Biology, Dept. of Biopharmaceutics, School of Pharmacy, Fourth Military Medical University, Xi’an 710032, China DOI 10.1002/btpr.2086 Published online 00 Month 2015 in Wiley Online Library (wileyonlinelibrary.com)
Human epidermal growth factor (hEGF) is a cellular factor that promotes cell proliferation and has been widely used for the treatment of wounds, corneal injuries, and gastric ulcers. Recombinant hEGF (rhEGF) has previously been expressed using the pTWIN1 system with pH-induced intein and a chitin-binding domain. The rhEGF protein can be purified by chitin affinity chromatography because of the high affinity between the chitinbinding domain fusion-tag and the column. However, uncontrolled cleavage presents a major problem with this method. To overcome this problem, a novel purification method has been developed for a pH-induced intein tag rhEGF that is expressed in Escherichia coli. Following purification by denaturation of inclusion bodies, the fusion protein is renatured and simultaneously induced to self-cleave by dialysis. Further purification of rhEGF is achieved by heat treatment and ion-exchange chromatography. Our results show that the purity of rhEGF obtained through this method is over 98% and the quantity of purified rhEGF is 248 mg from a 1 L culture or 2,967 mg from a 12 L culture. Therefore, we conclude that we have developed an efficient purification method of rhEGF, which may be used for the purification of other heat-resistant and acid-resistant recombiC 2015 American Institute of Chemical Engineers Biotechnol. Prog., nant proteins. V 000:000–000, 2015 Keywords: intein, rhEGF, heat treatment, ion-exchange chromatography
Introduction Epidermal growth factor (EGF) is a protein consisting of 53 amino acid residues and contains three disulfide bonds. It was first discovered in the submaxillary gland of the mouse.1 Human epidermal growth factor (hEGF) was first isolated from urine in 1975.2 Functional studies showed that hEGF causes cell proliferation and inhibits gastric acid secretion. As a result, hEGF became commonly used in healing wounds, corneal injuries, and gastric ulcers.3,4 Because of the increasing demand for hEGF for clinical applications, many studies have attempted to increase the production of rhEGF by recombinant DNA technology.5–8 Unfortunately, expression of small peptides in Escherichia coli is extremely ineffective because of rapid peptide degradation by intracellular proteases.5,9,10 Alternatively, small peptides fused with an affinity tag are stable and resistant to proteolytic degradation in E. coli. Moreover, the target protein can be easily Correspondence concerning this article should be addressed to L. Wang at [email protected] (or) Y. Wan at [email protected] C 2015 American Institute of Chemical Engineers V
purified by affinity chromatography. In the majority of cases, following purification, the affinity tag is removed by treatment with a site-specific protease, particularly for proteins destined for medical applications. However, there are some problems associated with the removal of affinity tags using a site-specific protease: (1) after enzymatic cleavage, residual amino acids from the protein tag may be immunogenic and can be particularly problematic for proteins destined for clinical use;11 (2) highly specific proteases, including endopeptidases, are typically expensive and can be cost-prohibitive unless purchased in bulk.12 The inteins are protein-splicing elements, which can selfexcise from their host protein and catalyze ligation of exteins together.13 The intein-tagged protein purification system (IMPACT-TWIN system, NEB) first reported by Chong et al. in 1997 is a system that utilizes the inducible self-cleavage activity of intein to separate the target protein from the affinity tag.14,15 The unique features of this affinity purification systems are as follows: first, the target fusion protein is separated in one step using affinity chromatography, e.g., chitin affinity chromatography (chitin-AFC); 1
2
Biotechnol. Prog., 2015, Vol. 00, No. 00
Table 1. Examples of pH-Induced Inteins for Protein Purification Self-Cleavage Conditions on Chitin Column Target Protein Class 1 protein (PorA) Basic human fibroblast growth factor Human b-defensin 2 Human epidermal growth factor (hEGF) Human insulin Antimicrobial peptides
Scale
T ( C)
pH
Shake flask Fermentation Shake flask Shake flask Shake flask Shake flask
4 23 25 37 25 22
7.0 6.5 4.0–6.0 6.0 6.5 6.5
Time 5 days 16 h 24 h 24 h 2 days 2 days
Yield (mg/L)
Refs.
8.89 800 4 18 3.5 12.3
16 17 18 19 20 21
In this study, an optimized method for pH-induced, inteinmediated purifications of rhEGF is described. The rhEGF protein is fused with the Ssp dnaB intein at the N-terminus, and the construct was expressed in the E. coli inclusion body. The inclusion bodies were renatured, and Ssp dnaB intein self-cleavage was simultaneously induced by dialysis with pH shift at 4 C. Further purification of rhEGF was performed by heat treatment and IEC, and the resulting protein was over 98% pure. The approach described here may be useful for the purification of other heat-resistant and acidresistant recombinant proteins.
Materials and Methods Materials
Figure 1. Schematic representation of the pTWIN1-rhEGF plasmid.
second, the target protein can be released from the affinity tag in the absence of protease; third, no extra amino acid residues are introduced into the released target protein, which prevents any unexpected immunogenicity in clinical applications. In previous studies (Table 1), chitin-AFC had been successfully used in the pH-induced intein-mediated purification of the Class 1 protein (PorA),16 basic human fibroblast growth factor,17 human b-defensin 2,18 hEGF,19 human insulin,20 and antimicrobial peptides.21 However, to date, almost all of the proteins produced have been limited to small-scale purifications because of uncontrolled cleavage, which is a significant limitation in pH-induced inteins, such as Ssp dnaB derived from the Synechocystis sp dnaB gene and DICM derived from the Mtu RecA gene.12,17,21 The unexpected cleavage can be observed throughout chitin resin and resulted in the major source of loss. In addition, although the chitin resin can interact with the chitin-binding domain (CBD) tag efficiently, the capacity of chitin resin is 2 mg/mL and can be regenerated only five times using a low flow rate (95% pure by Bandscan 5.0 analysis. Two purification schemes for rhEGF were reported in the literature by Esipov et al.19: (1) recombinant rhEGF with reduced cysteines was bound to a chitin column, cleaved, eluted, and rhEGF was refolded to allow the formation of the proper cysteine bridges (Figure 7A); and (2) the entire
Figure 6.
Chromatogram of ion-exchange chromatography for purification of rhEGF.
fusion protein was refolded to form the proper disulfide bonds, then loaded onto the affinity resin, and rhEGF was cleaved and eluted in its native state (Figure 7B). The difference between the two schemes is the order of renaturation in the procedure. The study by Esipov et al. indicated that CBD-Ssp dnaB-rhEGF can be purified by dilution renaturation, and chitin-AFC. However, in our preliminary experiments, when CBD-Ssp DnaB-rhEGF was refolded by dialysis, rhEGF was released before intein self-cleavage was activated by lowering the pH. Previous studies have also shown that premature cleavage is a significant limitation of pH-induced intein formation.12 To overcome this obstacle, we performed intein-mediated cleavage concurrently with dialysis in this study. We adopted a gradient renaturation and cleavage strategy during dialysis by controlling buffer pH through the exchange of the refolding and cleavage buffers to obtain a mixed solution of uncleaved protein (CBDSsp dnaB-rhEGF), fusion tag (CBD-Ssp dnaB), and rhEGF. Heat treatment and IEC were used in place of chitin-AFC. DEAE-Sepharose FF resin has high capacity of protein and can be used on a large scale. The improved purification procedure is shown in Figure 7C. The yield and purity after each purification step are summarized in Table 2. Recently, commercially produced rhEGF has been purified by expanded-bed adsorption, trypsin digestion, and cation exchange chromatography to a final yield of 223 mg/L.8 The process has been scaled up and commercialized by Bharat Biotech (Hyderabad, India). In comparison, the yield of rhEGF obtained in our study was 248 mg/L (Table 2), which is comparable with the yield obtained by Bharat Biotech, indicating that our method may be suitable for the commercial production of rhEGF.
Scaling-up production of rhEGF Our purification process using denaturing solution was scaled up from 250 mL to 3 L in a pilot plant. Refolding and tag cleavage were performed by dialysis against refolding buffer and cleavage buffer in the low-temperature chromatography refrigerator (Shanghai Xinyu Equipment Co., Shanghai, China), with about 3.5 g target protein released from the fusion protein. After heat treatment, rhEGF was separated from the fusion tag by centrifugation, and any remaining fusion tag was removed by IEC with a column (80 mm 3 300 mm i.d) containing 1 L DEAE-Sepharose FF resin. Approximately, 3 g rhEGF at over 98% purity was
6
Biotechnol. Prog., 2015, Vol. 00, No. 00
Figure 7. Comparison of purification schemes for pH-induced, intein-mediated rhEGF from E. coli inclusion bodies. A: purification scheme I by chitin resin; B: purification scheme II by chitin resin; C: purification scheme by heat treatment and IEC. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com]
Table 2. Comparison of Purification Yield and Purity of rhEGF at Small and Pilot Scales Small Scale in the Laboratory (1 L)
Large scale in pilot plant (12 L)
Purification Steps
Total Protein (mg)*
Target Protein (mg)†
Purity (%)‡
Yield (%)
Total Protein (mg)*
Target Protein (mg)†
Purity (%)‡
Yield (%)
Inclusion bodies
3,347
74
100§
40,500
100§
816
36
60.1
9,760
29,716 (CBD-Ssp dnaB-rhEGF) 3,518 (rhEGF)
74
Renaturation and cleavage supernatant Heat treatment IEC
2,478 (CBD-Ssp dnaB-rhEGF) 294 (rhEGF)
36
59.9
266 253
250 (rhEGF) 248 (rhEGF)
94 98
51.1 50.7
3,190 3,020
2,986 (rhEGF) 2,967 (rhEGF)
93.5 98
50.9 50.7
*Total protein was estimated from Coomassie blue-stained SDS-PAGE gel using BSA as a standard. The rhEGF protein was calculated based on the formula: target protein 5 total protein 3 approximate purity. ‡ The purities of protein were determined by Bandscan 5.0 analysis of the gel. § The 100% yield indicated the amount of rhEGF in the inclusion bodies. †
Figure 8. Characterization of the purified rhEGF. (A) HPRPLC analysis of the purity of rhEGF. (B) MALDI-TOF-MS analysis of the molecular weight of rhEGF. The determined molecular weight was 6,224.01 Da.
Biotechnol. Prog., 2015, Vol. 00, No. 00
obtained in the pilot plant without any obvious decrease in yield (Table 2). Characterization and biological activity of rhEGF HPRPLC of the final purified rhEGF (Figure 8A) showed that the purity of the final product is higher than 98%. Analysis of antibody binding capacity by enzyme-linked immunosorbent assay showed that rhEGF is recognized by an antiEGF monoclonal antibody. The molecular weight of the rhEGF was 6.2 kDa by MALDI-TOF-MS (Figure 8B), which is identical to its theoretical value. We used an MTT assay to determine the biological activity of rhEGF, and our results indicate enhanced cell proliferation after treatment with 40 ng/mL rhEGF. The measured rate of cell proliferation is 1.7 times higher than that of the negative control, with P-value < 0.05. Compared with standard rhEGF, the activity of the purified rhEGF is 5 3 106 IU/mg.
Conclusions In summary, we have developed an improved purification method to prepare pH-inducible, intein-mediated rhEGF. In contrast to previous methods, an essential feature of this method is that dialysis can be used to induce self-cleavage of the intein, and heat treatment and IEC were used in place of chitin-AFC to purify rhEGF. As a result, we minimized target protein loss due to uncontrolled cleavage. Based on SDSPAGE and HPRPLC analysis, the purity of rhEGF obtained after purification was above 98%. Analysis of the yield from each purification step indicates that inefficient cleavage is a major source of loss of rhEGF. Therefore, further studies should be performed to overcome this technical challenge so that this technique may be applied to commercial hEGF production. We hope that this method may be useful for the purification of other heat-resistant and acid-resistant recombinant proteins in a more cost-effective and productive manner.
Acknowledgments This work was supported by the Science Foundation of Shaanxi Academy of Sciences (Nos. 2013K-07 and 2014K-06) and the Foundation of Science and Technology in Shaanxi Province (Nos. 2010K12-01-05 and 2014K11-02-02-01).
Literature Cited 1. Cohen S. Isolation of a mouse submaxillary gland protein accelerating incisor eruption and eyelid opening in the new-born animal. J Biol Chem. 1962; 237:1555–1562. 2. Starkey RH, Cohen S, Orth DN. Epidermal growth factor: identification of a new hormone in human urine. Science. 1975; 189: 800–802. 3. Cohen S, Carpenter G. Human epidermal growth factor: isolation and chemical and biological properties. Proc Natl Acad Sci U S A. 1975; 72:1317–1321. 4. Huang BR, Cai LW, Wang X. Purification of recombinant hEGF expressed in yeast Pichia pastoris and the study on its characters. Zhongguo Yi Xue Ke Xue Yuan Xue Bao. 2001; 23: 106–110. 5. Allen G, Paynter CA, Winther MD. Production of epidermal growth factor in Escherichia coli from a synthetic gene. J Cell Sci Suppl. 1985; 3:29–38.
7 6. Lee JY, Yoon CS, Chung IY, Lee YS, Lee EK. Scale-up process for expression and renaturation of recombinant human epidermal growth factor from Escherichia coli inclusion bodies. Biotechnol Appl Biochem. 2000; 31(Pt 3):245–248. 7. Lee YS, Suh CW, Park SK, Lee EK. Purification of soluble human epidermal growth factor (hEGF) from recombinant Escherichia coli culture broth by using expanded-bed adsorption chromatography. Biotechnol Appl Biochem. 2003; 38:9–13. 8. Sharma K, Babu PV, Sasidhar P, Srinivas VK, Mohan VK, Krishna E. Recombinant human epidermal growth factor inclusion body solubilization and refolding at large scale using expanded-bed adsorption chromatography from Escherichia coli. Protein Expr Purif. 2008; 60:7–14. 9. Skipper N, Sutherland M, Davies RW, Kilburn D, Miller RC Jr, Warren A, Wong R. Secretion of a bacterial cellulase by yeast. Science. 1985; 230:958–960. 10. Abdull Razis AF, Ismail EN, Hambali Z, Abdullah MN, Ali AM, Mohd Lila MA. The periplasmic expression of recombinant human epidermal growth factor (hEGF) in Escherichia coli. Asia Pac J Mol Biol Biotechnol. 2006; 14:41–45. 11. Makrides SC. Strategies for achieving high-level expression of genes in Escherichia coli. Microbiol Rev. 1996; 60:512–538. 12. Fong BA, Wu WY, Wood DW. The potential role of selfcleaving purification tags in commercial-scale processes. Trends Biotechnol. 2010; 28:272–279. 13. Elleuche S, Poggeler S. Inteins, valuable genetic elements in molecular biology and biotechnology. Appl Microbiol Biotechnol. 2010; 87:479–489. 14. Chong S, Mersha FB, Comb DG, Scott ME, Landry D, Vence LM, Perler FB, Benner J, Kucera RB, Hirvonen CA, Pelletier JJ, Paulus H, Xu MQ. Single-column purification of free recombinant proteins using a self-cleavable affinity tag derived from a protein splicing element. Gene. 1997; 192:271– 281. 15. New England Biolabs. IMPACTTM. CN Instruction Manual. Ipswich, MA: New England Biolabs; 2004. 16. Humphries HE, Christodoulides M, Heckels JE. Expression of the class 1 outer-membrane protein of Neisseria meningitidis in Escherichia coli and purification using a self-cleavable affinity tag. Protein Expr Purif. 2002; 26:243–248. 17. Sharma SS, Chong S, Harcum SW. Intein-mediated protein purification of fusion proteins expressed under high-cell density conditions in E. coli. J Biotechnol. 2006; 125:48–56. 18. Diao H, Guo C, Lin D, Zhang Y. Intein-mediated expression is an effective approach in the study of beta-defensins. Biochem Biophys Res Commun. 2007; 357:840–846. 19. Esipov RS, Stepanenko VN, Chupova LA, Boyarskikh UA, Filipenko ML, Miroshnikov AI. Production of recombinant human epidermal growth factor using ssp dnaB mini-intein system. Protein Expr Purif. 2008; 61:1–6. 20. Chen T, Li L, Hao H, Qiao Y. Preparation of monomeric b27 lys destripeptide insulin by intein mediated expression in Escherichia coli. Protein Expr Purif. 2011; 80:152–156. 21. Wang H, Meng XL, Xu JP, Wang J, Wang H, Ma CW. Production, purification, and characterization of the cecropin from Plutella xylostella, pxCECA1, using an intein-induced selfcleavable system in Escherichia coli. Appl Microbiol Biotechnol. 2012; 94:1031–1039. 22. Tada H, Shiho O, Kuroshima K, Koyama M, Tsukamoto K. An improved colorimetric assay for interleukin 2. J Immunol Methods. 1986; 93:157–165. 23. Abdull Razis AF, Ismail EN, Hambali Z, Abdullah MN, Ali AM, Mohd Lila MA. Expression of recombinant human epidermal growth factor in Escherichia coli and characterization of its biological activity. Appl Biochem Biotechnol. 2008; 144:249– 261. 24. Carpenter G, Cohen S. Epidermal growth factor. Annu Rev Biochem. 1979; 48:193–216. Manuscript received Apr. 21, 2014, and revision received Feb. 3, 2015.
Lili Wang Key Laboratory of Synthetic and Natural Functional Molecule Chemistry of Ministry of Education, Shaanxi Key Laboratory of Modern Separation Science, Institute of Modern Separation Science, Northwest University, Xi’an 710069, China
Xiaochang Xue State Key Laboratory of Cancer Biology, Dept. of Biopharmaceutics, School of Pharmacy, Fourth Military Medical University, Xi’an 710032, China DOI 10.1002/btpr.2086 Published online 00 Month 2015 in Wiley Online Library (wileyonlinelibrary.com)
Human epidermal growth factor (hEGF) is a cellular factor that promotes cell proliferation and has been widely used for the treatment of wounds, corneal injuries, and gastric ulcers. Recombinant hEGF (rhEGF) has previously been expressed using the pTWIN1 system with pH-induced intein and a chitin-binding domain. The rhEGF protein can be purified by chitin affinity chromatography because of the high affinity between the chitinbinding domain fusion-tag and the column. However, uncontrolled cleavage presents a major problem with this method. To overcome this problem, a novel purification method has been developed for a pH-induced intein tag rhEGF that is expressed in Escherichia coli. Following purification by denaturation of inclusion bodies, the fusion protein is renatured and simultaneously induced to self-cleave by dialysis. Further purification of rhEGF is achieved by heat treatment and ion-exchange chromatography. Our results show that the purity of rhEGF obtained through this method is over 98% and the quantity of purified rhEGF is 248 mg from a 1 L culture or 2,967 mg from a 12 L culture. Therefore, we conclude that we have developed an efficient purification method of rhEGF, which may be used for the purification of other heat-resistant and acid-resistant recombiC 2015 American Institute of Chemical Engineers Biotechnol. Prog., nant proteins. V 000:000–000, 2015 Keywords: intein, rhEGF, heat treatment, ion-exchange chromatography
Introduction Epidermal growth factor (EGF) is a protein consisting of 53 amino acid residues and contains three disulfide bonds. It was first discovered in the submaxillary gland of the mouse.1 Human epidermal growth factor (hEGF) was first isolated from urine in 1975.2 Functional studies showed that hEGF causes cell proliferation and inhibits gastric acid secretion. As a result, hEGF became commonly used in healing wounds, corneal injuries, and gastric ulcers.3,4 Because of the increasing demand for hEGF for clinical applications, many studies have attempted to increase the production of rhEGF by recombinant DNA technology.5–8 Unfortunately, expression of small peptides in Escherichia coli is extremely ineffective because of rapid peptide degradation by intracellular proteases.5,9,10 Alternatively, small peptides fused with an affinity tag are stable and resistant to proteolytic degradation in E. coli. Moreover, the target protein can be easily Correspondence concerning this article should be addressed to L. Wang at [email protected] (or) Y. Wan at [email protected] C 2015 American Institute of Chemical Engineers V
purified by affinity chromatography. In the majority of cases, following purification, the affinity tag is removed by treatment with a site-specific protease, particularly for proteins destined for medical applications. However, there are some problems associated with the removal of affinity tags using a site-specific protease: (1) after enzymatic cleavage, residual amino acids from the protein tag may be immunogenic and can be particularly problematic for proteins destined for clinical use;11 (2) highly specific proteases, including endopeptidases, are typically expensive and can be cost-prohibitive unless purchased in bulk.12 The inteins are protein-splicing elements, which can selfexcise from their host protein and catalyze ligation of exteins together.13 The intein-tagged protein purification system (IMPACT-TWIN system, NEB) first reported by Chong et al. in 1997 is a system that utilizes the inducible self-cleavage activity of intein to separate the target protein from the affinity tag.14,15 The unique features of this affinity purification systems are as follows: first, the target fusion protein is separated in one step using affinity chromatography, e.g., chitin affinity chromatography (chitin-AFC); 1
2
Biotechnol. Prog., 2015, Vol. 00, No. 00
Table 1. Examples of pH-Induced Inteins for Protein Purification Self-Cleavage Conditions on Chitin Column Target Protein Class 1 protein (PorA) Basic human fibroblast growth factor Human b-defensin 2 Human epidermal growth factor (hEGF) Human insulin Antimicrobial peptides
Scale
T ( C)
pH
Shake flask Fermentation Shake flask Shake flask Shake flask Shake flask
4 23 25 37 25 22
7.0 6.5 4.0–6.0 6.0 6.5 6.5
Time 5 days 16 h 24 h 24 h 2 days 2 days
Yield (mg/L)
Refs.
8.89 800 4 18 3.5 12.3
16 17 18 19 20 21
In this study, an optimized method for pH-induced, inteinmediated purifications of rhEGF is described. The rhEGF protein is fused with the Ssp dnaB intein at the N-terminus, and the construct was expressed in the E. coli inclusion body. The inclusion bodies were renatured, and Ssp dnaB intein self-cleavage was simultaneously induced by dialysis with pH shift at 4 C. Further purification of rhEGF was performed by heat treatment and IEC, and the resulting protein was over 98% pure. The approach described here may be useful for the purification of other heat-resistant and acidresistant recombinant proteins.
Materials and Methods Materials
Figure 1. Schematic representation of the pTWIN1-rhEGF plasmid.
second, the target protein can be released from the affinity tag in the absence of protease; third, no extra amino acid residues are introduced into the released target protein, which prevents any unexpected immunogenicity in clinical applications. In previous studies (Table 1), chitin-AFC had been successfully used in the pH-induced intein-mediated purification of the Class 1 protein (PorA),16 basic human fibroblast growth factor,17 human b-defensin 2,18 hEGF,19 human insulin,20 and antimicrobial peptides.21 However, to date, almost all of the proteins produced have been limited to small-scale purifications because of uncontrolled cleavage, which is a significant limitation in pH-induced inteins, such as Ssp dnaB derived from the Synechocystis sp dnaB gene and DICM derived from the Mtu RecA gene.12,17,21 The unexpected cleavage can be observed throughout chitin resin and resulted in the major source of loss. In addition, although the chitin resin can interact with the chitin-binding domain (CBD) tag efficiently, the capacity of chitin resin is 2 mg/mL and can be regenerated only five times using a low flow rate (95% pure by Bandscan 5.0 analysis. Two purification schemes for rhEGF were reported in the literature by Esipov et al.19: (1) recombinant rhEGF with reduced cysteines was bound to a chitin column, cleaved, eluted, and rhEGF was refolded to allow the formation of the proper cysteine bridges (Figure 7A); and (2) the entire
Figure 6.
Chromatogram of ion-exchange chromatography for purification of rhEGF.
fusion protein was refolded to form the proper disulfide bonds, then loaded onto the affinity resin, and rhEGF was cleaved and eluted in its native state (Figure 7B). The difference between the two schemes is the order of renaturation in the procedure. The study by Esipov et al. indicated that CBD-Ssp dnaB-rhEGF can be purified by dilution renaturation, and chitin-AFC. However, in our preliminary experiments, when CBD-Ssp DnaB-rhEGF was refolded by dialysis, rhEGF was released before intein self-cleavage was activated by lowering the pH. Previous studies have also shown that premature cleavage is a significant limitation of pH-induced intein formation.12 To overcome this obstacle, we performed intein-mediated cleavage concurrently with dialysis in this study. We adopted a gradient renaturation and cleavage strategy during dialysis by controlling buffer pH through the exchange of the refolding and cleavage buffers to obtain a mixed solution of uncleaved protein (CBDSsp dnaB-rhEGF), fusion tag (CBD-Ssp dnaB), and rhEGF. Heat treatment and IEC were used in place of chitin-AFC. DEAE-Sepharose FF resin has high capacity of protein and can be used on a large scale. The improved purification procedure is shown in Figure 7C. The yield and purity after each purification step are summarized in Table 2. Recently, commercially produced rhEGF has been purified by expanded-bed adsorption, trypsin digestion, and cation exchange chromatography to a final yield of 223 mg/L.8 The process has been scaled up and commercialized by Bharat Biotech (Hyderabad, India). In comparison, the yield of rhEGF obtained in our study was 248 mg/L (Table 2), which is comparable with the yield obtained by Bharat Biotech, indicating that our method may be suitable for the commercial production of rhEGF.
Scaling-up production of rhEGF Our purification process using denaturing solution was scaled up from 250 mL to 3 L in a pilot plant. Refolding and tag cleavage were performed by dialysis against refolding buffer and cleavage buffer in the low-temperature chromatography refrigerator (Shanghai Xinyu Equipment Co., Shanghai, China), with about 3.5 g target protein released from the fusion protein. After heat treatment, rhEGF was separated from the fusion tag by centrifugation, and any remaining fusion tag was removed by IEC with a column (80 mm 3 300 mm i.d) containing 1 L DEAE-Sepharose FF resin. Approximately, 3 g rhEGF at over 98% purity was
6
Biotechnol. Prog., 2015, Vol. 00, No. 00
Figure 7. Comparison of purification schemes for pH-induced, intein-mediated rhEGF from E. coli inclusion bodies. A: purification scheme I by chitin resin; B: purification scheme II by chitin resin; C: purification scheme by heat treatment and IEC. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com]
Table 2. Comparison of Purification Yield and Purity of rhEGF at Small and Pilot Scales Small Scale in the Laboratory (1 L)
Large scale in pilot plant (12 L)
Purification Steps
Total Protein (mg)*
Target Protein (mg)†
Purity (%)‡
Yield (%)
Total Protein (mg)*
Target Protein (mg)†
Purity (%)‡
Yield (%)
Inclusion bodies
3,347
74
100§
40,500
100§
816
36
60.1
9,760
29,716 (CBD-Ssp dnaB-rhEGF) 3,518 (rhEGF)
74
Renaturation and cleavage supernatant Heat treatment IEC
2,478 (CBD-Ssp dnaB-rhEGF) 294 (rhEGF)
36
59.9
266 253
250 (rhEGF) 248 (rhEGF)
94 98
51.1 50.7
3,190 3,020
2,986 (rhEGF) 2,967 (rhEGF)
93.5 98
50.9 50.7
*Total protein was estimated from Coomassie blue-stained SDS-PAGE gel using BSA as a standard. The rhEGF protein was calculated based on the formula: target protein 5 total protein 3 approximate purity. ‡ The purities of protein were determined by Bandscan 5.0 analysis of the gel. § The 100% yield indicated the amount of rhEGF in the inclusion bodies. †
Figure 8. Characterization of the purified rhEGF. (A) HPRPLC analysis of the purity of rhEGF. (B) MALDI-TOF-MS analysis of the molecular weight of rhEGF. The determined molecular weight was 6,224.01 Da.
Biotechnol. Prog., 2015, Vol. 00, No. 00
obtained in the pilot plant without any obvious decrease in yield (Table 2). Characterization and biological activity of rhEGF HPRPLC of the final purified rhEGF (Figure 8A) showed that the purity of the final product is higher than 98%. Analysis of antibody binding capacity by enzyme-linked immunosorbent assay showed that rhEGF is recognized by an antiEGF monoclonal antibody. The molecular weight of the rhEGF was 6.2 kDa by MALDI-TOF-MS (Figure 8B), which is identical to its theoretical value. We used an MTT assay to determine the biological activity of rhEGF, and our results indicate enhanced cell proliferation after treatment with 40 ng/mL rhEGF. The measured rate of cell proliferation is 1.7 times higher than that of the negative control, with P-value < 0.05. Compared with standard rhEGF, the activity of the purified rhEGF is 5 3 106 IU/mg.
Conclusions In summary, we have developed an improved purification method to prepare pH-inducible, intein-mediated rhEGF. In contrast to previous methods, an essential feature of this method is that dialysis can be used to induce self-cleavage of the intein, and heat treatment and IEC were used in place of chitin-AFC to purify rhEGF. As a result, we minimized target protein loss due to uncontrolled cleavage. Based on SDSPAGE and HPRPLC analysis, the purity of rhEGF obtained after purification was above 98%. Analysis of the yield from each purification step indicates that inefficient cleavage is a major source of loss of rhEGF. Therefore, further studies should be performed to overcome this technical challenge so that this technique may be applied to commercial hEGF production. We hope that this method may be useful for the purification of other heat-resistant and acid-resistant recombinant proteins in a more cost-effective and productive manner.
Acknowledgments This work was supported by the Science Foundation of Shaanxi Academy of Sciences (Nos. 2013K-07 and 2014K-06) and the Foundation of Science and Technology in Shaanxi Province (Nos. 2010K12-01-05 and 2014K11-02-02-01).
Literature Cited 1. Cohen S. Isolation of a mouse submaxillary gland protein accelerating incisor eruption and eyelid opening in the new-born animal. J Biol Chem. 1962; 237:1555–1562. 2. Starkey RH, Cohen S, Orth DN. Epidermal growth factor: identification of a new hormone in human urine. Science. 1975; 189: 800–802. 3. Cohen S, Carpenter G. Human epidermal growth factor: isolation and chemical and biological properties. Proc Natl Acad Sci U S A. 1975; 72:1317–1321. 4. Huang BR, Cai LW, Wang X. Purification of recombinant hEGF expressed in yeast Pichia pastoris and the study on its characters. Zhongguo Yi Xue Ke Xue Yuan Xue Bao. 2001; 23: 106–110. 5. Allen G, Paynter CA, Winther MD. Production of epidermal growth factor in Escherichia coli from a synthetic gene. J Cell Sci Suppl. 1985; 3:29–38.
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