title: [Construction of expression plasmids harbouring genes encoding recombinant FN polypeptides with triple-domain and preliminary characterization of the products expressed in Escherichia coli].
abstract: 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.
authors.affiliation: Department of Medical Molecular Biology, Tongji Medical University, Wuhan.
authors.forename: M C
authors.initials: MC
authors.lastname: Li
authors.forename: Z H
authors.initials: ZH
authors.lastname: Feng
authors.forename: D
authors.initials: D
authors.lastname: Li
authors.forename: G M
authors.initials: GM
authors.lastname: Zang
mesh_terms: D000818:Animals; D004926:Escherichia coli / Q000235:genetics*; D005353:Fibronectins / Q000096:biosynthesis* / Q000627:therapeutic use; D008546:Melanoma, Experimental / Q000188:drug therapy; D009362:Neoplasm Metastasis / Q000517:prevention & control; D010957:Plasmids; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000627:therapeutic use
chemical_list: D005353:Fibronectins; D011994:Recombinant Proteins