An improved procedure for production of human epidermal growth factor from recombinant E. coli.
An improved procedure for the fermentation and purification of human epidermal growth factor (hEGF) was developed. Recombinant Escherichia coli HB-101 [lacUV5omp08hEGF] harboring plasmid lacUV5omp08hEGF encoding hEGF was used in fermentation to increase levels of hEGF. Medium composition, and the levels of inoculum, inducer (isopropyl-beta-D-thiogalactoside) and ampicillin were optimized with respect to volumetric fermentation of hEGF. As a result, the hEGF concentration reached a high value of 242 mg l(-1) and the amount of heterogeneous protein decreased by 62% compared with that before optimization in batch fermentation. High-quality hEGF was purified from the fermentation culture by centrifugation, salting-out, resuspension, recentrifugation and finally gel chromatography on a Grad-iFrac System using Sephadex G-50 superfine. The purity of hEGF and the total yield were more than 94% and higher than 36%, respectively, and SDS-PAGE of the purified hEGF demonstrated a single band corresponding to an hEGF standard. In particular, a very important phenomenon was found, i.e. that the amount of heterogenous protein in fermentation broths cultured in media with high concentrations of lactose is far less than that cultured in media with high concentrations of glucose.
Tong
W Y
WY
Department of Chemical and Biochemical Engineering, Zhejiang University, Hangzhou, People's Republic of China.
Yao
S J
SJ
Zhu
Z Q
ZQ
Yu
J
J
D001709:Biotechnology; D003470:Culture Media; D004815:Epidermal Growth Factor / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D004926:Escherichia coli / Q000235:genetics / Q000254:growth & development / Q000378:metabolism; D005285:Fermentation; D006801:Humans; D066298:In Vitro Techniques; D007785:Lactose / Q000378:metabolism; D008970:Molecular Weight; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification
D003470:Culture Media; D011994:Recombinant Proteins; D004815:Epidermal Growth Factor; D007785:Lactose