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Scale‐up process for expression and renaturation of recombinant human epidermal growth factor from Escherichia coli inclusion bodies
Author(s) -
Lee Jong Yeon,
Yoon Chang Shin,
Chung Il Yup,
Lee Young Seek,
Lee Eun Kyu
Publication year - 2000
Publication title -
biotechnology and applied biochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.468
H-Index - 70
eISSN - 1470-8744
pISSN - 0885-4513
DOI - 10.1042/ba19990101
Subject(s) - recombinant dna , inclusion bodies , complementary dna , epidermal growth factor , escherichia coli , chemistry , biochemistry , monomer , expression vector , cystine , microbiology and biotechnology , cysteine , biology , chromatography , gene , enzyme , organic chemistry , receptor , polymer
A cDNA encoding mature epidermal growth factor (EGF) was isolated and cloned into a pQE30 vector in which the His 6 ‐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.