High-level Expression And Purijfication Of Recombinant Human Insulin-like Growth Factor-1in Escherichia Coli | Posted on:2014-11-13 | Degree:Master | Type:Thesis | Country:China | Candidate:C Zong | Full Text:PDF | GTID:2250330428463046 | Subject:Biochemical Engineering | Abstract/Summary: | PDF Full Text Request | IGF-1(Insulin-like growth factor-1) can regulate glucose metabolism in the cells. Also IGF-1can be used as one growth factor for animal cell culture. Since the concentration of IGF-1is rather low in animal serum, it is very expensive to produce IGF-1directly from animal serum, and there are some potential risks of contamination due to its animal orgins. In the present work, gene technology is adopted to produce bioactive IGF-1in recombinant E.coli.Two redundant amino acids sequence introduced by pET-32a(+) vector were eliminated from N-terminal of hIGF-1gene through site-directed mutagenesis of the plasmid. Then the expression conditions in this E. coli Rosetta (DE3)/pET32-hIGF-M host were systematically investigated. After the optimization of the expression conditions, the expression level of soluble hIGF-1fusion protein was greatly improved. The optimal expression conditions were in the following:when the culture was effectively induced at the middle exponential phase with0.6mM IPTG for6hours at34℃. The highest productivity of soluble hIGF-1fusion protein (1.84g/L) was achieved.After cell disruption, the soluble fusion protein was separated effectively through affinity chromatography and cleaved by enterokinase. The recovery of hIGF-1fusion protein is80%with high purity (94%). Finally mature hIGF-1with90%purity was obtained from enterokinase digestion of the purifed hIGF-1fusion with the aide of one more affinity chromatography.Moreover, batch culture and fed-batch culture of recombinant cells were compared. The highest expression level (11.7g/L) was achieved by a glycerol-based high cell-density cultivation strategy in a10-L fermentor. The effect of mature hIGF-1on the growth of NIH/3T3fibroblasts was examined. The results showed that the quanity of living cells was increased by30percent by introduing hIGF-1sample with regard to those in the control group. | Keywords/Search Tags: | insulin-like growth factor-1, Expression, affinity chromatography, highcell density culture | PDF Full Text Request | Related items |
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