|
Insulin -like growth factor (insulin-like growth factors, referred to IGFs), also known as insulin-like growth factor, is a kind of multifunctional regulator of cell proliferation . Insulin -like growth factor system consists of IGF-Ⅰ and IGF-Ⅱ, Ⅰ and type Ⅱ insulin-like growth factor receptor (insulin-like growthfactor receptor, IGFR) as well as with IGF -specific binding of insulin-like growth factor binding protein (insulin like growth factor binding protein, IGFBP) family composed of cell proliferation in normal and malignant transformation processes play an important role . The insulin-like growth factor binding protein (IGFBP) is a transport and storage form of IGF , IGF binding proteins usually along with the production and secretion , and thus for the regulation of IGFBP concentrations of free IGF and its receptors play an important role . Have been found in 10 kinds of insulin-like growth factor binding protein (IGFBP1-10) in IGFBP-4 is the smallest molecular weight , is an inhibitor of cell proliferation , originally from the human osteoblast conditioned medium was purified out , IGFBP-4 main functions : regulation of IGF role in particular cellular matrix stored in IGF, IGF independent play a role. In order to obtain a large number of high purity IGFBP-4, reducing its production costs , to study the in vivo protein function and the regulation of IGFBP-4 basis . Using recombinant protein expression is to get a lot of the main way , this study will use chemical synthesis method IGFBP-4 gene fragments were amplified by PCR of its , and build support with the target gene is connected , insert the pGEX-6p- a fusion protein expression vector, the recombinant plasmid pGEX/-6p-1-IGFBP-4. After transformed into E. coli BL21, high efficiency screening transformants , shake flasks , the constructed IGFBP-4 expression plasmid by IPTG (isopropyl-l-thio-beta-D-galactoside) induced expression , collecting cell lysis , SDS- PAGE electrophoresis test results show that , IGFBP-4 expression successfully . Further induction time , temperature and pH of the medium induced optimized detected by SDS-PAGE to determine the optimum conditions for the expression of 16 ℃, pH9.5, 20h, 0.2mM IPTG, the purpose of this condition supernatant total protein the highest , indicating that the IGFBP-4 expression system in E. coli was highly expressed . The resulting fusion protein can be isolated by affinity chromatography purification.
|