Prokaryotic Expression Of Human Epidermal Growth Factor (hEGF)

Since Cohen firstly discovered epidermal growth factor(EGF) from a mouse in 1960, many growth factors have identified one after another mainly from human and mammilla after 40 years such as transforming growth factorα(TGFα),vaccinia growth factor(VGF),Shope fibroma growth factor(SFGF),myxoma growth factor(MGF),ampiregulin,arnphireguli(AR),betacellulin(BTC) ,epiregulin(EPl) ,heparin-binging EGF-like growth factor(HB-EGF),vaccinia virus growth factor(VVGF) ect. They have 50-80 amino acids and homology about 20%~90% in primary structure. In their molecules there have conservatively six cysteines, which form three disulfide bonds, make their bind to EGF receptor inducing similar biological activity. These molecules are defined as an EGF family.Human epidermal growth factor is a small single-chain polypeptide of 53 amino acids. It has heat-stable and acid-stable. It's main biology effects are to stimulate the growth and proliferation of epidermal and epithelial cell both in vivo and in vitro. It is often applied to treat burns, scald, cornea transplant, gastric acid, duodenal ulcered and also use as cosmetic. It has big latency economic value because it has special and potential biological effects.Human EGF gene was successfully amplified from human placenta with RT-PCR method, then cloned it into expression vector: pGEX-4T-1 and pGEX-6P-1 and transformed them into BL21-CodonPlus?–RIL and BL21-CodonPlus?–RP, which don't need added program to solve the coding of rare codons. The fusion hEGF was about 30% of total protein measured with SDS-PAGE. The expression product is mainly inclusion bodies. Collecting and dissolving of bodies was renaturation with reduced Glu. Separating and purifing the inclusion bodies and obtained fusion hEGF has biological activity then passing through Glutathione SepharoseTM 4B. Western blotting test identified that the separated and purificated protein is objective protein hEGF. Because hEGF is a kind of content of cell culture medium without blood serum, using addition the GST-hEGF fusion protein to cell culture medium of EP, it has been shown the fusion proteins have good biological activity.