Studies On Fermentation Of The Recombinant Human Insulin-like Growth Factor I Engineering Bacterial And Purification Of Recombinant Protein

Insulin-like growth factor I ( IGF-I ) is a multifunctional cell regulating factor. Recombinant human insulin-like growth factor I (rhIGF-I ) appears promising to be developed as a drug in the treatment of many diseases. It is possible to produce it on a large scale by means of modern biotechnology. It is an indispensable technology for the fermentation to produce gene engineering drugs on a large scale. We established recombinant hIGF-I engineering bacteria Escherichia co/i BL2 1 (DE3) /pRSET-TGF, observed its growth and explored the rules of recombinant protein expression. On the base of above studies, we processed the pilot study of high density fermentation. The final cell density (0D600) reached 20, and the recombinant protein expression level was 25.4% of the total cellular protein. A series of preliminary fermentation technics of recombinant hIGF-I engineering bacteria had been formed. Similar to majority of the eukaryotic proteins, rhIGF-I was expressed as inclusion bodies in E. co/i. A series of purification steps, including cell breakage, inclusion body washing, inclusion body solubilization, protein renaturation and ion-exchange chromatography, have been set up to purify the recombinant protein in an active form. Experimental results showed that 67.8% purity of inclusion body could obtained due to inclusion body washing with 0.05%Triton X-l00 and lmol/L urea; the inclusion body solubility increased with increasing urea concentration. Stepwise decrease of urea concentration in the buffer could effectively improve the protein renaturation efficiency by reducing protein aggregation. At the same time, reduced and oxidized glutathionine were added to optimize the correct disulfide bonds formation.The recombinant protein was then purified by Q Sepherose F F ion-exchange chromatogiaphy. The final protein recovery was 23.49%, and the purity was 85.27%. Mass spectrometry assay revealed the molecular weight of objective protein was 7.8K]). Western blotting indicated that recombinant protein could react with antibodies against anti-IGF-I. The effect of the stimulating Nll-13T3 cell proliferation showed that rhIGF-J had some biological activity.