| Objective: p93 and Nelin ,two cardiac-specific expression genes, were isolated based on large-scale ESTs(expressed sequence tags) sequencing from adult heart cDNA library recently. Prokaryotic expression vector of p93 and Nelin including full-length and/or deletants have been constructed by molecular cloning technology. In order to gain the recombinant proteins, the expressed and purified of two genes have been carried futher.Methods: (1) The GST fusion expression vectors pGEX-5X-p93 incuding full-length cDNA of p93 was transformed into Escherichia coli strain BL21, induced with isopropyl-B-D-thiogala-ctopyranoside (IPTG). purified with Glutathione Sepharose-4B and SDS-PAGE respectively. (2) p93 and C-terminal fragment of p93 with Nco \l Xho I restriction sites,(the last named p93-C) was amplified from PGEX-5X- p93 plasmid containing the full-length of p93 gene by PCR. The PCR products and plasmid pET28a(+) were digested by corresponding restriction endonucleases respectively.The fragments were ligated by T4 DNA ligase to gain the recombinant expression vector pET28a-p93 and pET28a-p93-C. The vectors were identified by endonuclease digesting and the fragments of p93 and p93-C that inserted into the vector were confirmed by DNA sequencing. Meanwhile,another recombinant expression vectors pET28a-p93 and pET28a-p93-C earring His tag were transformed into Escherichia coli strain BL21(DE3), The expression of transformants was analyzed by SDS-PAGE after induced with IPTG, p93-his6 and p93-C-his6 fusion protein were purified with Ni2+ metal chelate affinity chromatography columns. (3) The GST-p93 fusion protein was analysed by anti-GST polyclonal antibody, p93-hisf, and p93-C-his6 proteins were determined by anti-GST-p93 polyclonal antibody using Western blot hybridization. (4) Predicting of B cell epitopes of human Nelin: hydrophilicity plot , surface probability plot , antigenic index and flexibility were obtained by the methods of Kyte-Doolittle , Emini,Welling and Karplus-Schulz, respectively, combining the results according to these methods to predict the B cell epitopes of human Nelin. According to the predicting B cell epitopes, F-actin binding domain and Ig domain, the deletants of Nelin-l(l-133aa), Nelin-2(66-252 aa) and Nelin-3(227-448aa) were designed. (5) The prokaryotic expression vector pGEX-5X- Nelin-l~3 and pET28a-Nelin-l were constrcted by molecular cloning technology, the expressed Nelin-1 -hiss protein was purified with Ni2+ metal chelate affinity chromatography columns. The expressed proteins were verified using Western blot.Results: (1 )GST-p93 fusion protein was overexpressed as inclusion bodies, the GST-p93 fusion protein purified with Glutathione Sepharose-4B contained partly bacteria protein, the proteins can be purified to 95 % relative homogeneity using the method of preparative gel polyacrylamide gel electrophoresis. (2) Endonuclease digesting and the fragments of p93 and p93-C confirmed by DNA sequencing were correctly inserted into the vector. Both p93-C-his6 and p93-his6 were expressed as inclusion bodies. The purity of p93-C-his6 and p93-hise fusion protein were over 95% purified by Ni + metal chelate affinity chromatography . The yields of p93-C-his6 and p93-his() were 2 mg and 0.5mg per 100ml culture respectively. (3) The expressed protein of pGEX-5X-p93, pET28a-p93 and pET28a-p93-C were correctly products confirmed with Western blot. (4) The fragmats of Nelin N-terminal No. 27-39aa. 90-102aa and 158-170aa may be the predominant epitopes of the B cells. (5) The expression amount of Nelin-1 was higher than Nelin-2 and Nelin-3. Purified Nelin-1-hisr, deletant protein with the N-terminal 27-39aa and 90-102aa of Nelin gene was obtained. The purity was over 95% by SDS-PAGE.Conclusion: The purification of GST-p93 and Nelin-1 - hise lay a foundation for preparation of antibodies to p93 and Nelin gene.The expression and purification of p93 and p93-C protein in E.coli made it possible to study further on refolding and biological function of p93.
|
PDF Full Text Request