Prokaryotic expression and purification of Tiam1 truncated proteins / 医学研究生学报

ABSTRACT

Objective Tiam1, a member of guanine nucleotide exchange factors, plays an important role in tumor invasion and metastasis.This study aimed to construct Tiam1 truncated recombinant plasmids and induce the expression of GST-tagged human Tiam1 fusion proteins in Escherichia coli (E.coli), followed by purification and identification of the GST-Tiam1 fusion protein.Methods The cDNA fragments of Tiam1 C685, C751 and C1199 were amplified by PCR and cloned into the pGEX-4T-1 vector.After verified by DNA sequencing, the recombinant plasmid was transformed into E.coli BL21 (DE3) competent cells, and the expression of the fusion protein was induced by IPTG.The GST-tagged human Tiam1 fusion proteins were purified with glutathione-agarose resin and indentified by Western blot.Results Three Tiam1 truncated recombinant plasmids were constructed successfully.The recombinant fusion proteins GST-Tiam1 C685, GST-Tiam1 C751, and GST-Tiam1 C1199 were expressed mainly in the form of soluble proteins in the cell lysate supernatant with expected relative molecular weight of 100, 108, and 157 kD.Conclusion The recombinant plasmids expressing the bioactive fusion proteins were constructed successfully, which has prepared the ground for the subsequent studies of the Tiam1 protein.