Construction of Expression Plasmids Harbouring Genes Encoding Recombinant FN Polypeptides with Triple-domain and Preliminary Characterization of the Products Expressed in Escherichia coli
三结构域重组FN多肽表达质粒的构建及其表达产物性质的初步鉴定

To investigate the important role of recombinant triple-domain FN polypeptide in tumor therapy, two expression plasmids pF94-62 and pF94-82 were constructed and used to express triple-domain polypeptides of human FN in E. coli. The expressed polypeptides were CH62 (Pro 1239-Ser 1515 of FN linked with Ala 1690-Val 2049 through Met) and CH82 (CH62 without Pro 1953-Glu 1978). CH82 polypeptide was expressed as inclusion bodies in E. coli cultured at 37 degrees C. After denaturation with 8 mol/L urea and renaturation, the polypeptides were purified by the affinity chromatograph with Heparin-agarose, and the purified product was analysed by cell adhesion assay. The expression level of CH62 in E. coli was very low(5%), but that of CH82 was very high (21%), it suggested that N terminal sequence of Cell II in FN was the key sequence which influence the expression of triple-domain polypeptide in E. coli. The purified product was capable of binding heparin and cells, and it had a better binding activity than bifunctional-domain FN polypeptides. The production of CH82 polypeptide provided a fundamental basis for further study of recombinant product with better function of anti-metastasis and immune regulation.