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Biotechnology and Applied Biochemistry (2000) 31, (245–248) (Printed in Great Britain)

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

Jong Yeon Lee*, Chang Shin Yoon†, Il Yup Chung†, Young Seek Lee† and Eun Kyu Lee*¹

*Department of Chemical Engineering, Hanyang University, Ansan, Korea 425-791, and †Department of Biochemistry and Molecular Biology, Hanyang University, Ansan, Korea 425-791

Abbreviations used: EGF, epidermal growth factor; rhEGF, recombinant human EGF; IB, inclusion bodies; IPTG, isopropyl b-D-thiogalactoside; RP-HPLC, reversed-phase HPLC; Ni²⁺-NTA, Ni²⁺-nitrilotriacetate; TFA, trifluoroacetic acid.

¹ To whom correspondence should be addressed.

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 b-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.

Received 8 November 1999/22 February 2000; accepted 8 March 2000

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