Scale-up process for expression and renaturation of recombinant human epidermal growth factor from Escherichia coli inclusion bodies

• Published in: Biotechnology and applied biochemistry Vol. 31; no. 3; pp. 245 - 248
• Main Authors: Lee, Jong Yeon, Yoon, Chang Shin, Chung, Il Yup, Lee, Young Seek, Lee, Eun Kyu
• Format: Journal Article
• Language: English
• Published: Oxford, UK Blackwell Publishing Ltd 01-06-2000; Portland Press
• Subjects: b-D-thiogalactoside; Biological and medical sciences; Biotechnology; Cell Division - drug effects; Cell Line - drug effects; Culture Media; epidermal growth factor; Epidermal Growth Factor - chemistry; Epidermal Growth Factor - genetics; Epidermal Growth Factor - metabolism; Epidermal Growth Factor - pharmacology; Escherichia coli; Escherichia coli - genetics; fed-batch; Fundamental and applied biological sciences. Psychology; Health. Pharmaceutical industry; Hormones. Growth factors; Humans; Inclusion Bodies - genetics; Industrial applications and implications. Economical aspects; l-cystine; Oxidation-Reduction; Production of active biomolecules; Protein Renaturation; recombinant EGF; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Recombinant Proteins - pharmacology; refolding; Human; Escherichia coli; Gene expression; Biological activity; Dimensioning; Denaturation renaturation; Epidermal growth factor; Production; Microorganism culture; Bacteria; Inclusion body; Recombinant protein; Semicontinuous; Enterobacteriaceae
• ISSN: 0885-4513; 1470-8744
• DOI: 10.1042/BA19990101

A cDNA encoding mature epidermal growth factor (EGF) was isolated and cloned into a pQE30 vector in which the His6‐tagged EGF was expressed. pH‐stat feeding of concentrated medium at the time of isopropyl β‐d‐thiogalactoside induction and slug‐feedings of the enriched medium during the induction resulted in a higher cell density and specific expression. Using a simple refolding protocol that consisted of 1 mM l‐cysteine addition for a 1‐h reduction followed by 5 mM l‐cystine addition for oxidative refolding, we were able to convert nearly all EGF monomers into the oxidized form. Also, the refolding aggregate was converted into the monomeric form. Approx. 50% overall yield was obtained from the dissolved inclusion bodies to a single peak under FPLC. We hope that the result of this study may provide information that is useful for the scale‐up of the recombinant human EGF production process.