Abstract：ObjectiveTo establish a prokaryotic expression system of the breast cancer marker gene C35 for breast carcinoma diagnosis.Wild type and C112S mutant of C35 prokaryotic recombinants were constructed and expressed the in E.coli strain BL21,and two kinds of C35 proteins were purified.MethodsSequences encoding wild type and C112S(open reading frame) of C35 were amplified by PCR using breast cancer cell line T47D cDNA, The PCR products were cloned into the BamHⅠand XhoI sites of the vector pGLO1.The positive recombinants of pGLO1-C35(wild type) and pGLO1-C35(C112S) were identified by double-digesting and sequencing,and were overexpressed in E.coli strain BL21,respectively.To optimize protein purification conditions,10mL of bacteria were incubated in lactose broth at 37℃ to an absorbance (A600) of 0.8,following different time gradients(3h,6h and 9h) with 1 mM isopropyl-D-1-thiogalactopyranoside（IPTG） at different temperature such as 37℃,22℃ and 15℃.After the cells that carried C35 recombinants were induced by the optimized conditions and harvested,the generated bacteria were suspended in resuspension buffer and lysed by sonication,the supernatants were loaded onto the Ni2+ Chelating Sepharose Fast Flow column for affinity chromatography of the N-terminal 6×His tagged wild type or C112S C35 proteins.Finally,the wild type and C112S mutant of C35 proteins were identified by Western blot and quantified by ultramicro-spectrophotometer under 280 nm.ResultsThe double-digesting and sequencing results indicated that both wild type and C112S mutant of C35 ORFs were successfully inserted into pGLO1 between BamHⅠand XhoI sites.Based on the time and temperature selections,the optimized conditions were identified as inducing with 1 mM IPTG for 4-6h at 37℃,and high amount of C35 proteins could be expressed under these conditions.Both wild type and mutant C35 proteins were expressed soluble and chelated on Ni2+ sepharose beads with high affinity.Pure wild type and mutant C35 proteins were confirmed by SDS-PAGE and Western blot,indicating that the purified wild type and mutant C35 protein had high purity and fine immune activity.The final yield of purified wild type and mutant C35 proteins were about 1.5mg/mL with a purity of about 90%.ConclusionsThe prokaryotic expression systems of wild type and mutant C35 gene are successfully established.The wild type and C112S mutant of C35 proteins with high purity and concentration could be yielded under the optimal expression conditions identified in this paper.Our work may be useful for developing breast carcinoma prophase diagnosis kit based on C35 antiserum.
