Clone, Expression And Purification Of Insulin-like Growth Factor Binding Protein-4 (IGFBP-4) Gene

Insulin-like growth factors are a series of multi-functional cell proliferation regulatory factor. Insulin-like growth factor system is composed by IGF-Ⅰand IGF-Ⅱ,ⅠandⅡinsulin-like growth factor receptor(IGFR),as well as the IGF specificity union's insulin-like growth factor binding protein(IGFBP) family,which play an important role in process of hyperplasia and malignant transformation in the normal cell.And insulin-like growth factor binding protein(IGFBP) is transportation and storage form of IGF,IGF usually produces and secretes together with binding protein,thus IGFBP plays an important role in regulating the concentration of free IGF and with receptor integration.In the present time,it has been found 10 kinds of insulin-like growth factor binding protein (IGFBP1-10).IGFBP-4 is the smallest molecular mass,is an inhibiting factor of cell proliferation, initially separated and purified from the osteoblasts in the conditioned medium.The main roles of IGFBP-4:regulating IGF,storing IGF in the specific cell matrixes,playing the role without relying on IGF.In order to obtain a large number of high purity IGFBP-4,to lower its production costs,we studied function of IGFBP-4 protein and laid the foundation for the IGFBP-4 regulation and control mechanism in vivo.Using the gene recombination expression obtains the massive goal protein,this study will use chemical method to synthesize the IGFBP-4 gene fragment,amplify by PCR,and build the gene vector in order to connect with goal gene,and insert pGEX-6p-1 fusion protein expression vector,build the recombinant plasmid pGEX/-6p-1-IGFBP-4.After transformed host bacteria BL21,screened the high-efficiency conversion factor,cultivated in shake flask,the IGFBP-4 expression plasmid was constructed.The plasmid was induced and expressed by IPTG(isopropyl-1-thio-beta-D-galactoside IPTG),collected cell lysates,these results showed that IGFBP-4 expression was successful using SDS-PAGE gel electrophoresis.To further optimize the induction time,the induction temperature and the medium pH,identified the most suitable expression condition for 16℃,pH9.5,20h,0.2mM IPTG by SDS-PAGE examination, under the conditions,the expression total amount of the supernatant goal protein was the maximum,showed that IGFBP-4 gained the highly effective expression in E.coli expression system.Fusion protein can be obtained by affinity chromatography to separate and purify.