Eukaryotic Expression and Identification of the Recombination Protein of Rat Decay-accelerating Factor with Pichia pastoris Expression System

WANG Wei, ZHANG Li, ZHANG Peng

Abstract

To construct yeast eukaryotic expression vector carrying the gene of rat decay-accelerating factor (DAF), and to induce the expression of recombinant protein. Methods The cDNA of rat DAF was amplified by RT-PCR from the fresh hepatic tissue of rat. The PCR product was inserted into the Pichia pastoris vector pPIC9K. Then the recombinant plasmid pPIC9K-DAF was transformed into yeast GS115 through electroporation. The positive high copy number transformants were rapidly selected by using G418-YPD and were induced by methanol. The induced product was analyzed by DNA sequencing, SDS-PAGE and Western blot. Finally, the bio-activity of recombinant rat DAF was examined. Results The rat DAF cDNA was successfully cloned in pPIC9K. The positive clone in pPIC9K expression vector (pPIC9K-DAF) was sieved, and the pPIC9K-DAF showed the same seqencing result as reported in GenBank. And the recombination protein of rat DAF (relative molecular mass is 70×103) has natural rat immunogenicity and bio-activity. Conclusion Successfully cloning and high-level expression of rat DAF recombination protein in Pichia pastoris laid a foundation for further research on immunity and complement system.

 

Keywords: Pichia pastoris, Eukaryotic expression, pPIC9K, Decay-accelerating factor

 

 

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References


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