Construction Of DTMP-HSA D? And DTMP-HSA Recombinant Vectors And Their Transient Expression In CHO-S Cells

Previousley,our laboratory has constructed dTMP-HSA recombinant protein expression vectors,and expressed the dTMP-HSA fusion proteins by yeast and CHO-S cells.The transient expression by CHO-S cells was 30-40 mg/L,while the stable expression was 300-500 mg/L.The bioactivity in vitro of dTMP-HSA fusion protein expressed by CHO-S cells was 50,000-80,000 U/mg,ahout 100 folds of that of yeast.But it is only 17-27%of the positive drug rhTPO,still at a low level.To increase the activity of TMP fusion protein,we fused the tanderm TPO mimetic peptide?dTMP?with the HSA domain??HSA D??and the constructed recombinant vector dTMP-HSA D?was expressed by yeast.Results revealed that the bioactivity in vitro of the truncated fusion propein dTMP-HSA D?is 10-20 folds of its full length conuterpart dTMP-HSA.For the purpose of improving the activity further,we constructed two dTMP-HSA D?recombinant protein expression vectors:pcDNA3/15-3D₃F and pCHO1.0/15-3D₃F with the mammalian cell expression vectors pcDNA3 and pCHO1.0.To make comparison with pcDNA3/15-3D₃F,the full length of HSA fusion protein expression vector pcDNA3/15-3F was constructed at the same time.The signal peptide of recombinant vectors constructed above is all of Fc signal peptide.To study the effect of signal peptides on the expression of recombinant proteins,we replaced the Fc signal peptide in the constructs mentioned above with HSA signal peptide,and constructed pcDNA3/15-3H,pcDNA3/15-3D₃H,pCHO1.0/15-3H,pCHO1.0/p15-3D₃H recombinant vectors.All recombinant protein expression vectors constructed above were expressed in CHO-S cells.WB assay indicated that both of dTMP-HSA D?and dTMP-HSA fuison proteins contained the TMP domain and HSA D?or HSA domain,confirming the structure is integrated.The double-antibody sandwich ELISA quantification assay displayed that the expression of dTMP-HSA D?fusion protein is 0.4-0.9 mg/L,while the dTMP-HSA is 30-35 mg/L.Therein,the expression of the dTMP-HSA recombinant vector pcDNA3/15-3F,pCHO1.0/15-3F,pcDNA3/15-3H,pCHO1.0/15-3H was 35 mg/L,30 mg/L,11 mg/L,8 mg/L,respectively.While the expression of the dTMP-HSA D?recombinant vector pcDNA3/15-3D₃F and pCHO1.0/15-3D₃F was 0.9 mg/L,0.4 mg/L respectively.Neither of concentrations of the fusion proteins expressed by plasmid pcDNA3/15-3D₃H and pCHO1.0/15-3D₃H was determined.Obviously,for the transient expression of dTMP-HSA D?and dTMP-HSA recombinant vectors in CHO-S cells,the expression of Fc signal peptide is more advantageous than HSA signal peptide,and the pcDNA3 vector is better than pCHO1.0 vector,and the full length dTMP-HSA fusion protein has higher expression level than that of the truncated dTMP-HSA D?.The in vitro bioactivity detection assay performed using Dual-Glo^TM Luciferase Assay System confirmed that the bioactivity of dTMP-HSA fuion protein is50,000⁸0,000 U/mg,the truncated dTMP-HSA D?is 700,000-100,0000 U/mg,10-20 times of the full length protein dTMP-HSA and 2-3 times of the positive drug rhTPO.The physical&chemical property and three-dimension?3D?structure was analyzed by on line tools.It indicated that the in vivo serum half-life of dTMP-HSA D?and dTMP-HSA are both 30 h,but the functional group on the sturture of dTMP-HSA D?is more favour of its interaction with the TPO receptor c-Mpl,which ensure its higher bioactivity than the full length protein dTMP-HSA.In a word,compared to the full length protein dTMP-HSA and rhTPO,the activity of the truncated dTMP-HSA D?fusion protein has prominently improved,and our study is beneificial to the study of improving the activity of TMP fusion proteins.