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Economic optimization of expression of soluble human epidermal growth factor in Escherichia coli

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Abstract

Human epidermal growth factor (hEGF) has multiple biological functions, such as promoting cell proliferation, differentiation, and migration. In addition, it is a very expensive polypeptide with attractive market prospects. However, the production of hEGF needs for high cost to manufacture polypeptide demands reinvestigations of process conditions so as to enhance economic benefits. Improving the expression of soluble hEGF is the fundamental method to reduce the cost. In this study, a non-extracellular engineered strain of expressed hEGF was constructed, using plasmid pET-22b(+) in Escherichia coli. Preliminary fermentation and high cell density cultivation were carried out in shake flasks and in a 5 L bioreactor, respectively. A high yield of 98 ± 10 mg/L of soluble hEGF and a dry cell weight (DCW) of 6.98 ± 0.3 g/L were achieved in shake flasks. Then, fermentation conditions were optimized for large-scale production, while taking into consideration the expensive equipment required for cooling and conforming to industrial standards. A yield of 285 ± 10 mg/L of soluble hEGF, a final cell density of 57.4 ± 2 g/L DCW (OD600 141.1 ± 4.9), and hEGF productivity of 14.3 mg/L/h were obtained using a bioreactor at 32 °C for 20 h. The production method developed in this study for the biosynthesis of soluble hEGF is efficient and inexpensive.

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References

  • Abdull Razis AF, Ismail EN, Hambali Z, Abdullah MNH, Ali AM, Mohd Lila MA (2008) Expression of recombinant human epidermal growth factor in Escherichia coli and characterization of its biological activity. Appl Biochem Biotechnol 144:249–261

    Article  PubMed  CAS  Google Scholar 

  • Baeshen NA, Baeshen MN, Sheikh A, Bora RS, Ahmed MM, Ramadan HA, Saini KS, Redwan EM (2014) Cell factories for insulin production. Microb Cell Fact 13:141

    Article  PubMed  PubMed Central  Google Scholar 

  • Baneyx F (1999) Recombinant protein expression in Escherichia coli. Curr Opin Biotechnol 10:411–421

    Article  PubMed  CAS  Google Scholar 

  • Bhoria S, Yadav J, Yadav H, Chaudhary D, Jaiwal R, Jaiwal PK (2022) Current advances and future prospects in production of recombinant insulin and other proteins to treat diabetes mellitus. Biotechnol Lett 44:643

    Article  PubMed  CAS  Google Scholar 

  • Bordeaux M, de Girval D, Rullaud R, Subileau M, Dubreucq E, Drone J (2014) High-cell-density cultivation of recombinant Escherichia coli, purification and characterization of a self-sufficient biosynthetic octane ω-hydroxylase. Appl Microbiol Biotechnol 98:6275–6283

    PubMed  CAS  Google Scholar 

  • Carlson ED, Gan R, Hodgman CE, Jewett MC (2012) Cell-free protein synthesis: applications come of age. Biotechnol Adv 30:1185–1194

    Article  PubMed  CAS  Google Scholar 

  • Chen J, Li H, Chen J (2017) Human epidermal growth factor coupled to different structural classes of cell penetrating peptides: a comparative study. Int J Biol Macromol 105:336

    Article  PubMed  CAS  Google Scholar 

  • Chen X, Song B, Liu J, Yang J, He T, Lin Y, Zhang H, Xie C (2012) Modulation of gene expression in cold-induced sweetening resistant potato species Solanum berthaultii exposed to low temperature. Mol Genet Genomics 287:411–421

    Article  PubMed  CAS  Google Scholar 

  • Clare JJ, Romanos MA, Rayment FB, Rowedder JE, Smith MA, Payne MM, Sreekrishna K, Henwood CA (1991) Production of mouse epidermal growth factor in yeast: high-level secretion using Pichia pastoris strains containing multiple gene copies. Gene 105:205–212

    Article  PubMed  CAS  Google Scholar 

  • Clark ED (2001) Protein refolding for industrial processes. Curr Opin Biotechnol 12:202–207

    Article  PubMed  CAS  Google Scholar 

  • Cohen S (1965) The stimulation of epidermal proliferation by a specific protein (EGF). Dev Biol 12:394

    Article  PubMed  CAS  Google Scholar 

  • Cohen S, Carpenter G (1975) Human epidermal growth factor: isolation and chemical and biological properties. Proc Natl Acad Sci USA 72:1317–1321

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Cooke RM, Wilkinson AJ, Baron M, Pastore A, Tappin MJ, Campbell ID, Gregory H, Sheard B (1987) The solution structure of human epidermal growth factor. Nature 327:339–341

    Article  PubMed  CAS  Google Scholar 

  • D’Amore T, Celotto G, Russell I, Stewart GG (1989) Selection and optimization of yeast suitable for ethanol production at 40 °C. Enzyme Microb Technol 11:411–416

    Article  CAS  Google Scholar 

  • de Groot NS, Ventura S (2006) Effect of temperature on protein quality in bacterial inclusion bodies. FEBS Lett 580:6471–6476

    Article  PubMed  Google Scholar 

  • Desai PN, Shrivastava N, Padh H (2010) Production of heterologous proteins in plants: strategies for optimal expression. Biotechnol Adv 28:427–435

    Article  PubMed  CAS  Google Scholar 

  • Eissazadeh S, Moeini H, Dezfouli MG, Heidary S, Nelofer R, Abdullah MP (2017) Production of recombinant human epidermal growth factor in Pichia pastoris. Braz J Microbiol 48:286–293

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Eryasar-Orer K, Karasu-Yalcin S (2021) Optimization of activated charcoal detoxification and concentration of chestnut shell hydrolysate for xylitol production. Biotechnol Lett 43:1195–1209

    Article  PubMed  CAS  Google Scholar 

  • Esquirol Caussa J, Herrero Vila E (2015) Epidermal growth factor, innovation and safety. Med Clin-Barcelona 145:305–312

    Article  Google Scholar 

  • Ferreira RDG, Azzoni AR, Freitas S (2018) Techno-economic analysis of the industrial production of a low-cost enzyme using E. coli: the case of recombinant β-glucosidase. Biotechnol Biofuels 11:1–13

    Article  Google Scholar 

  • Gauglitz GG, Korting HC, Pavicic T, Ruzicka T, Jeschke MG (2011) Hypertrophic scarring and keloids: pathomechanisms and current and emerging treatment strategies. Mol Med 17:113–125

    Article  PubMed  CAS  Google Scholar 

  • Goeddel DV, Kleid DG, Bolivar F, Heyneker HL, Yansura DG, Crea R, Hirose T, Kraszewski A, Itakura K, Riggs AD (1979) Expression in Escherichia coli of chemically synthesized genes for human insulin. Proc Natl Acad Sci USA 76:106–110

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Gregory H (1975) Isolation and structure of urogastrone and its relationship to epidermal growth factor. Nature 257:325–327

    Article  PubMed  CAS  Google Scholar 

  • Gu Y, Lv X, Liu Y, Li J, Du G, Chen J, Rodrigo L, Liu L (2019) Synthetic redesign of central carbon and redox metabolism for high yield production of N-acetylglucosamine in Bacillus subtilis. Metab Eng 51:59–69

    Article  PubMed  Google Scholar 

  • Haider SR, Reid HJ, Sharp BL (2018) Tricine-SDS-PAGE. Springer, New York, pp 151–160

    Google Scholar 

  • Han G, Dong X, Zhang L, Fu L, Wang G, Sun Y (2012) Facilitated oxidative refolding of ribonuclease a from inclusion bodies with a new redox system. Biochem Eng J 69:106–112

    Article  CAS  Google Scholar 

  • Jang B, Ahn Y (2019) Enhanced recombinant insulin production in transgenic Escherichia coli that heterologously expresses carrot heat shock protein 70. Biocatal Agric Biotechnol 20:101180

    Article  Google Scholar 

  • Jia Y, Zhou J, Chang Y, An F, Li X, Xu X, Sun X, Xiong C, Wang J (2018) Effect of optimized concentrations of basic fibroblast growth factor and epidermal growth factor on proliferation of fibroblasts and expression of collagen. Chin Med J-Peking 131:2089–2096

    Article  CAS  Google Scholar 

  • Junker BH (2004) Scale-up methodologies for Escherichia coli and yeast fermentation processes. J Biosci Bioeng 97:347–364

    Article  PubMed  CAS  Google Scholar 

  • Kane JF, Hartley DL (1988) Formation of recombinant protein inclusion bodies in Escherichia coli. Trends Biotechnol 6:95–101

    Article  CAS  Google Scholar 

  • Le PU, Lenferink AEG, Pinard M, Baardsnes J, Massie B, Connor-Mccourt O (2009) Escherichia coli expression and refolding of e/k-coil-tagged EGF generates fully bioactive egf for diverse applications. Protein Expres Purif 64:108–117

    Article  CAS  Google Scholar 

  • Lecina M, Sarró E, Casablancas A, Gòdia F, Cairó JJ (2013) IPTG limitation avoids metabolic burden and acetic acid accumulation in induced fed-batch cultures of Escherichia coli m15 under glucose limiting conditions. Biochem Eng J 70:78–83

    Article  CAS  Google Scholar 

  • Lee D, Kuo T, Chen M, Tang T, Liu F, Weng C (2006) Expression of porcine epidermal growth factor in Pichia pastoris and its biology activity in early-weaned piglets. Life Sci 78:649–654

    Article  PubMed  CAS  Google Scholar 

  • Lin H, Lu P, Zhou M, Wu F, Weng L, Meng K, Yang D, Li S, Jiang C, Tian H (2019) Purification of recombinant human fibroblast growth factor 13 in E. coli and its molecular mechanism of mitogenesis. Appl Microbiol Biotechnol 103:7017–7027

    Article  PubMed  CAS  Google Scholar 

  • Lindner R, Moosmann A, Dietrich A, Böttinger H, Kontermann R, Siemann-Herzberg M (2014) Process development of periplasmatically produced single chain fragment variable against epidermal growth factor receptor in Escherichia coli. J Biotechnol 192:136–145

    Article  PubMed  CAS  Google Scholar 

  • Liu K, Li S, Pang X, Xu Z, Li D, Xu H (2017) Enhanced enzymatic synthesis of a cephalosporin, cefadroclor, in the presence of organic co-solvents. Appl Biochem Biotechnol 182:29–40

    Article  PubMed  CAS  Google Scholar 

  • Liu K, Zhang Y, Liu K, Zhao Y, Gao B, Tao X, Zhao M, Wang F, Wei D (2022) De novo design of a transcription factor for a progesterone biosensor. Biosens Bioelectron 203:113897

    Article  PubMed  CAS  Google Scholar 

  • Marston FA (1986) The purification of eukaryotic polypeptides synthesized in Escherichia coli. Biochem J 240:1–12

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Mei M, Zhou Y, Peng W, Yu C, Ma L, Zhang G, Yi L (2017) Application of modified yeast surface display technologies for non-antibody protein engineering. Microbiol Res 196:118–128

    Article  PubMed  CAS  Google Scholar 

  • Zheng Z, Yao S, Zhan X, Lin CC (2009) Improvement of hegf production with enhanced cell division ability using dissolved oxygen responses to pulse addition of tryptone. Biotechnol Bioproc E 14:52–59

    Article  CAS  Google Scholar 

  • Meyer DE, Chilkoti A (1999) Purification of recombinant proteins by fusion with thermally-responsive polypeptides. Nat Biotechnol 17:1112–1115

    Article  PubMed  CAS  Google Scholar 

  • Meynial-Salles I, Sophie D, Philippe S (2008) A new process for the continuous production of succinic acid from glucose at high yield, titer, and productivity. Biotechnol Bioeng 99:129–135

    Article  PubMed  CAS  Google Scholar 

  • Ogiso H, Ishitani R, Nureki O, Fukai S, Yamanaka M, Kim JH, Saito K, Sakamoto A, Inoue M, Shirouzu M, Yokoyama S (2002) Crystal structure of the complex of human epidermal growth factor and receptor extracellular domains. Cell 110:775–787

    Article  PubMed  CAS  Google Scholar 

  • Pinsach J, de Mas C, López-Santín J (2008) Induction strategies in fed-batch cultures for recombinant protein production in Escherichia coli: application to rhamnulose L-phosphate aldolase. Biochem Eng J 41:181–187

    Article  CAS  Google Scholar 

  • Qiao K, Wasylenko TM, Zhou K, Xu P, Stephanopoulos G (2017) Lipid production in Yarrowia lipolytica is maximized by engineering cytosolic redox metabolism. Nat Biotechnol 35:173–177

    Article  PubMed  CAS  Google Scholar 

  • Sajitha S, Vidya J, Varsha K, Binod P, Pandey A (2015) Cloning and expression of L-asparaginase from E. coli in eukaryotic expression system. Biochem Eng J 102:14–17

    Article  CAS  Google Scholar 

  • Shimizu N, Fukuzono S, Harada Y, Fujimori K, Gotoh K, Yamazaki Y (1991) Mass production of human epidermal growth factor using fed-batch cultures of recombinant Escherichia coli. Biotechnol Bioeng 38:37–42

    Article  PubMed  CAS  Google Scholar 

  • Singh SM, Panda AK (2005) Solubilization and refolding of bacterial inclusion body proteins. J Biosci Bioeng 99:303–310

    Article  PubMed  CAS  Google Scholar 

  • Singhvi P, Saneja A, Srichandan S, Panda AK (2020) Bacterial inclusion bodies: a treasure trove of bioactive proteins. Trends Biotechnol 38:474–486

    Article  PubMed  CAS  Google Scholar 

  • Sonntag MH, Ibach J, Nieto L, Verveer PJ, Brunsveld L (2014) Site-specific protection and dual labeling of human epidermal growth factor (hEGF) for targeting, imaging, and cargo delivery. Chem A Eur J 20:6019–6026

    Article  CAS  Google Scholar 

  • Sun W, Wang L, Liu H, Liu Y, Ren Y, Wang F, Wei D (2019) Characterization and engineering control of the effects of reactive oxygen species on the conversion of sterols to steroid synthons in Mycobacterium neoaurum. Metab Eng 56:97–110

    Article  PubMed  CAS  Google Scholar 

  • Thomas DR, Walmsley AM (2014) Improved expression of recombinant plant-made hEGF. Plant Cell Rep 33:1801–1814

    Article  PubMed  CAS  Google Scholar 

  • Türker M (2004) Development of biocalorimetry as a technique for process monitoring and control in technical scale fermentations. Thermochim Acta 419:73–81

    Article  Google Scholar 

  • Wang Y, Wang Z, Xu Q, Du G, Hua Z, Liu L, Li J, Chen J (2009) Lowering induction temperature for enhanced production of polygalacturonate lyase in recombinant Pichia pastoris. Process Biochem 44:949–954

    Article  CAS  Google Scholar 

  • Wong WKR, Huang ELRC, Wong RSC, Morris C, Hackett J (2001) Applications, and efficient large-scale production, of recombinant human epidermal growth factor. Biotechnol Genet Eng Rev 18:51–71

    Article  PubMed  CAS  Google Scholar 

  • Wong WKR, Ng KL, Lam CC, Hu XH, Lai NCY, Wang H, Sivakumar T (2017) Review article: reasons for underrating the potential of human epidermal growth factor in medical applications. J Anal Pharm Res 4:11–12

    Google Scholar 

  • Xu G, Zhu Q, Luo Y, Zhang X, Guo W, Dou W, Li H, Xu H, Zhang X, Xu Z (2015) Enhanced production of L-serine by deleting sdaa combined with modifying and overexpressing sera in a mutant of Corynebacterium glutamicum syps-062 from sucrose. Biochem Eng J 103:60–67

    Article  CAS  Google Scholar 

  • Yera-Alos IB, Alonso-Carbonell L, Valenzuela-Silva CM, Tuero-Iglesias AD, Moreira-Martinez M, Marrero-Rodriguez I, Lopez-Mola E, Lopez-Saura PA (2013) Active post-marketing surveillance of the intralesional administration of human recombinant epidermal growth factor in diabetic foot ulcers. BMC Pharmacol Toxicol 14:44

    Article  PubMed  PubMed Central  Google Scholar 

  • Zhang H, Li Z, Qian Y, Zhang Q, Du P, Gan R, Ye Q (2007) Cultivation of recombinant Escherichia coli for secretory production of human epidermal growth factor under control of PL promoter. Enzyme Microb Technol 40:708–715

    Article  CAS  Google Scholar 

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Acknowledgements

This work was supported by the National Nature Science Foundation of China (21776133), the Six Talent Peaks Project in Jiangsu Province (SWYY-027), and the National Natural Science Foundation of China (Grant No. 21776075).

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Authors and Affiliations

  1. State Key Laboratory of Bioreactor Engineering, Newworld Institute of Biotechnology, East China University of Science and Technology, 130 Meilong Road, Shanghai, 200237, China

    Kun Liu, Feng-Qing Wang, Bei Gao & Dongzhi Wei

  2. Anhui Engineering Laboratory for Industrial Microbiology Molecular Breeding, College of Biology and Food Engineering, Anhui Polytechnic University, Wuhu, 241000, China

    Kun Liu & Ming Zhao

  3. College of Food Science and Light Industry, Nanjing Tech University, No.30 South Puzhu Road, Pukou District, Nanjing, 211816, China

    Kun Liu & Hong Xu

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  1. Kun Liu
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  2. Feng-Qing Wang
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  3. Ming Zhao
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  4. Bei Gao
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  5. Hong Xu
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Liu, K., Wang, FQ., Zhao, M. et al. Economic optimization of expression of soluble human epidermal growth factor in Escherichia coli. Biotechnol Lett 44, 1401–1414 (2022). https://doi.org/10.1007/s10529-022-03308-0

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