Construction Of Dual Targeting Anti-breast Cancer Fusion Protein G129R-T3 Prokaryotic Expression Plasmid And Inducible Expression

Objective By gene cloning technique, the active fragment of tumstatin T3 and the hPRLR antagonist G129R were connected together to construct and purify the prokaryotic expression fusion protein G129R-T3, which can not noly inhibit the breast cell proliferation, but also suppress tumor angiogenes.Methods (1) The DNA coding sequence of Homo sapiens tumstatin gene (69-88 amino acid) with NdeⅠ(BamHⅠ) and XhoⅠrestricted enzyme recognition sequence at each end were amplified with three specific overlap complementary primers by overlap extension polymerase chain reaction (PCR) method. The PCR products were reclaimed and purified by polyacrylamide gel electrophoresis. (2) According to the cDNA sequence of Homo sapiens prolactin gene from the GeneBank, two specific primers were designed for PCR by using the primer 5.0 software. The DNA fragments of G129R with NdeⅠand XhoⅠ(BamHⅠ) restriction enzyme recognition sequence at each end were amplified, which use pET22b(+)G129R-G129R plasmid as the template. The products were reclaimed and purified by agarose gel electrophoresis. (3) To make use of the complementary end of BamHⅠrecognition sequence which located at the 3'end of G129R and the 5'end of T3, the recovered DNA fragments of G129R and T3 were mixed as templates, and the fusion DNA fragment of G129R-T3 was amplified by using the upstream primer of G129R and the downstream primer of T3 both of which have NdeⅠand XhoⅠrecognition sequence at each end. The products were reclaimed and purified by agarose gel electrophoresis. (4) The retrieved products were respectively cloned into pMD-18T vector, and the ligated products were transformed into E.coli DH5α. The plasmids extracted from the positive clones were digested with double restriction endonuclease NdeⅠand XhoⅠto obtain the target fragments. (5) The target fragments were inserted into the lineared vector pET22b(+) that was digested also by NdeⅠand XhoⅠrespectively to construct the prokaryotic expression vectors pET22b(+)-G129R-T3, pET22b(+)-G129R and pET22b(+)-T3, and then they were transformed into E.coli DH5α. After plasmids sequencing, the results were blasted in the nucleotide databases. Bioinformatics analysis was made by using the PrositeScan, ProtParam, TMHMM, HNN and SWISS-MODEL Workspace softwares. (6) Then the recombinant prokaryotic expression plasmids were transformed into Escherichia coli BL21(DE3) which was induced to express proteins byβ-D-thiogalactopyranoside (IPTG). The target proteins were obtained after harvest cells by centrifugation, cell extract preparation, solubilization, IDA His·Bind column equilibration, and protein elution. The supernatant and pellet of fusion protein were analyzed by SDS-PAGE and Western blot.Results The DNA fragments of G129R-T3, G129R and T3 were respectively amplified successfully by PCR, and they have the similar to the target fragments in gel electrophoresis. The retrieved products were respectively cloned into pMD-18T vectors, and then the recombinant plasmid vectors were digested by the restriction endonuclease NdeⅠand XhoⅠ. The target fragments were obtained and inserted into expression vector pET22b(+). After being detected by endonuclease digestion, and sequence results, we found each of them is 99% correct except two silent mutations. Bioinformatics analysis results suggest that G129R-T3 (G129R) may be an acidic, unstable and non-transmembrane protein. After transforming these recombinant expression plasmids into E.coli BL21(DE3), the inducing conditions were optimized. SDS-PAGE electrophoresis confirmed protein mainly exists in the form of insoluble inclusion bodies, which is located in the cytoplasm. Their molecular weights were about 27kD and 24kD respectively. The most effective inducing condition is to add 1mM IPTG to the E. coli BL21(DE3) at OD600 of 0.6～0.8, and then shake at 200rpm for approximately 4h at 37°C. Western blot analysis of the purified protein showed that G129R-T3 and G129R could be successfully expressed in E. coli BL21(DE3).Conclusions We have successfully constructed a dual targeting recombinant prokaryotic expression plasmid of pET22b(+)-G129R-T3 and expressed and purified it successfully from E.coli BL21(DE3). Further more, purified recombinant proteins were obtained through affinity chromatography, which lay a solid foundation for the further study of their pharmacological actions.