Studies On The Expression Of Several Growth Factors Fused With A Novel Collagen In E.coli

Collagen like protein Scl2 is a kind of proteins containing highly repetitive amino acid sequences.It is capable of resisting the degradation of a variety of proteases.Pure Scl2 can obtained through acid precipitation and enzymatic hydrolysis.As a fusion label,Scl2 have some advantages in purification.Scl2-M integrates several functional sites on the Scl2 sequence,which is highly soluble expression in E.coli(previously studied in our lab).Human basic fibrobl growth factor(hbFGF)has a high medical value in cell proliferation and differentiation,neovascularization,bone tissue development and the nervous system protection.With the development of research and application,more and more medical institutions began to use hbFGF for the treatment in disease such as nervous system,burns or make up,showing a broad,bright prospects.At present,hbFGF are expensive and can not meet the needs of large-scale applications.Nowadays,hbFGF are mainly expressed by the E.coli expression system.But some codons in the hbFGF gene are rare codons,resulting in low expression of non-fused hbFGF in E.coli.hbFGF also easily degraded by protease.In addition,hbFGF has four cysteine residues,easily formed intermolecular disulfide bond,resulting in the formation of dimers or even multimers.The main research content in this paper are listed as follows:First,the gene template pUC19-hbFGF was synthesized according to the codon preference of E.coli,and obtained the hbFGF gene by PCR amplification.The recombinant expression vector pET28a-Scl2-M-hbFGF was constructed,and the fusion protein Scl2-M-hbFGF was expressed in E.coli BL21(DE3).The expression conditions also investigated.The optimized expression conditions were as follows:in TB medium,when the bacteria growth to mid-logarithmic phase,0.1 mM IPTG was added and induced 10 hours at 30 ?.Under this condition,the soluble expression of Scl2-M-hbFGF was 0.85 g/L.Second,expressed the EK light chain,providing a digestion enzyme for purification.Then purified the fusion protein(Scl2-M-hbFGF)by acid precipitation instead of Ni2+affinity chromatography,the recovery of Scl2-M-hbFGF(>90%)was much higher than Ni2+ affinity chromatography(about 50%).Then purified hbFGF through enterokinase digestion and cation exchange chromatography,obtained high purity hbFGF.The bioactivity of hbFGF in promote cell proliferation was confirmed by MTT assay,and the number of cells was increased about 50%compared with the negative control.With glycerol as carbon source,the high density fermentation of E.coli BL21(DE3)/pET28a-Scl2-M-hbFGF was carried out on 5L fermentor by batch fermentation and fed-batch fermentation.A slightly higher yield of fusion protein was obtained through Fed batch fermentation,reaching 7.7g/L,and the yield of hbFGF is 2.5g/L in theory.The period of Fed batch fermentation is shorter than batch fermentation,more suitable for large-scale industrial applications.Finally,hEGF and IGF-1 were fused with Scl2-M and expressed,established a method for the efficient expression of small molecule proteins with Scl2-M as fusion protein.