Study On Improving The Activity And Expression Of TMP Fusion Protein

In recent years,the expression vectors of pPinka-11#(1-3),pPinka-15#(1-9)and pPinka-16#(1-9),which contained TMP and HSA gene had been constructed in our Laboratory.Furthermore,the recombinant protein had been got by transforming them into the Pichia pastoris and shaking the flask.Moreover,the amount of recombinant protein were about 1.25g/L.However,the biological activity of them was only(300-8000)U/mg,which had a long distance compared with the positive control.In this thesis,the connection order of TMP and HSA,the linkage peptide between the two TMP and HSA domains had been screened respectively after many Yeast and Mammalian cell expression vectors being constructed in order to improve the activity and expression of TMP fusion protein.And then,the engineering strains or cell strains which had stable expression of target protein had been constructed successfully by transforming or transfecting.In the yeast expression system,the results show that the TMP fusion protein which expressed by the clone TMP-HSA was higher than that of clone HSA-TMP;the biological activity of the clone that the linkage peptide between the two TMP was L1 was higher than that of L2;the connection order of the two TMP in the HSA-(TMP)2 was end to end;the activity of the target protein which was expressed by the recombinant strain that contained(TMP)2-HSA-D3 that using the HSA-D3 domain instead of full length HSA was 1478U/mg,which was significantly higher than(TMP)2-HAS that the value was 361U/mg.The biological activity of purpose protein expressed by pCHO(15-15-2)was 16500U/mg,which had a higher significance compared with the Yeast expression system.The research has a great reference value for improving the activity and expression of TMP fusion protein.