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[Fusion construction, prokaryotic expression and structure characteristics prediction of bimolecular thrombopoietin].

AIM
Design, construction, expression in E coli and protein characteristics prediction of bimolecular thrombopoietin (T-T) with more stability, efficiency, and lower toxicity.


METHODS
The expression vectors of TPO and T-T, pET32 a(+)/TPO and pET32 a (+)/T-T, had been constructed by molecular cloning methods. Then, they were expressed in host bacterium. Their products were identified by Western blot. The protein characteristics, such as second structure, antigenicity, hydrophilicity, flexibility and isoelectric point, were predicted by DS Gene and Protscale software.


RESULTS
The expressing vectors pET32a(+)/TPO and T-T were constituted correctly and expressed in origami (DE3), and their expression efficiency were more than 40 percent of total protein. T-T was identified correctly by Western blot. DS Gene and Protscale software predict the protein characteristics of TPO sequences in T-T molecule were no change, there was high flexibility in the linker domain. But two amino acids in T-T molecule have been mutated, and an insert fragment with 34 amino acids following the linker had antigenicity, hydrophilicity, and beta-sheet structure.


CONCLUSION
We have constructed correctly and expressed T-T with high level in E Coli. Protein characteristics prediction of T-T accords with our design.