Expression and characterization of truncated extracellular domain of human transforming growth factor-beta receptor typeⅡdomain in Escherichia coli

HAN Rui-jie; WANG Xiao-hua; ZUO Kui-yang; CHU Yan-hui; LI Gui-qin; LIU Hai-feng

doi:10.3969/j.issn.1001-9448.2018.08.003

Abstract：Objective To construct the prokaryotic expression vector containing the extracellular domain of trans -forming growth factor-beta receptor type Ⅱ(thTβRⅡ, so as to evaluate its expression and characteristics .Methods The gene fragment of thTβRⅡfrom recombinant vector pGEX -4T1-TβRⅡwas amplified by regular PCR with specific primers, and cloned into the prokaryotic expression vector pET 28a.The recombinant protein pET28a-thTβRⅡwere in-duced with IPTG, purified with Ni2+-IDA affinity chromatography and characterized with SDS -PAGE and MTT assay. Expression of TGFβ1, FN mRNA, FN and α-SMA were assessed by real -time RT-PCR and Western blotting.Re-sults The PCR product of thTβRⅡdigested with two restriction enzymes (NdeI and BamHI) was cloned into the expres-sion vector pET28a, and confirmed by PCR, restriction endonuclease digestion and DNA sequence .The optimal expres-sion of recombinant protein thT βRⅡwas induced with 0.8 mM IPTG at 37℃for 5 h.The high-level expressed protein in the form of inclusion bodies was refolded by dialysis refolding procedures and purified by Ni 2+-IDA affinity chromatog-raphy, identified by SDS-PAGE.The recombinant protein thTGF -βRⅡ, at concentrations of 150 μg/mL, could signif- icantly inhibit the proliferation of TGF -β1-induced LX-2 cells; and at concentrations of 200 μg/mL, could signifi-cantly reduce the expression level of TGF β1, FN mRNA and FN, α-SMA protein.Conclusion The pET28a-thTβRⅡexpression vector is constructed , and the production of active recombinant protein thT βRⅡcould inhibit the prolifera-tion of TGF-β1-induced LX -2 cells, reduce the mRNA and protein expression levels of fibrosis -related factors, which lays the foundation for exploring the effect of the recombinant protein thT βRⅡin vitro and in vivo.