Construction of Prokaryotic Expression Vector and Prokaryotic Expression,Purification of sCAR-PPAb

To obtain a fusion protein containing soluble Coxsackie-adenovirus receptor(sCAR) and the domain B of Pinellia pedatisecta agglutinin(PPAb),the expression vector pQE30-sCAR-PPAb is constructed by inserting domain B of PPA gene into pQE30-sCAR which has constructed and is identified by PCR and sequence analysis.Then an expression strain is selected after transformation of the recombined plasmid into E.coli M15,fusion protein with His-tag is efficiently expressed in the form of inclusion body after IPTG induction.The inclusion body is washed,dissolved in renatured by the gradual removal of urea via dialysis in solubilization buffer and purified by Ni+2 chelate affinity chromatography under renatured condition.SDS-PAGE analysis and Western blotting with His-probe(mouse monoclonal IgG) show that the fusion protein with a molecular weight of about 42 is purified.Ad-EGFP transduction of leukemia cell lines Kasumi-1 is increased by sCAR-PPAb fusion protein,indicating that the purified protein is confirmed to be of bioactivity.