Soluble Expression of Recombinant Apoptin Fused with Mutant Cell-penetrating Peptide

Objective A recombinant expression vector of mutant apoptin-HBDCK-pET28a was constructed to investigate whether the recombinant proteins were highly expressed in a soluble form in E.coli BL21(DE3) and tested the mutant cell-penetrating peptide(CPP) transduction activity in HeLa cells.Methods A PCR mutation approach was used for the expressions of mutated apoptin-HBDCKand EGFP-HBDCK,which Cys(C) in heparin-binding domain(HBD) was mutated into Lys(K),respectively.The mutated fusion proteins were expressed in E.coli BL21(DE3) and purified by Ni2+-NTA affinity chromatography.The transduction activity of the mutant CPP in HeLa cells was detected.Results Both restriction enzyme and sequencing analysis proved that the mutant recombinant plasmid apoptin-HBDCK-pET28a and EGFP-HBDCK-pET28a were constructed correctly.Both fusion proteins were expressed in a soluble form in E.coli BL21(DE3).After a 13h incubation of HeLa cell with EGFP-HBDCK,an intensive green fluorescence was observed in most cells.Conclusion The fusion proteins can be expressed in a soluble form after a CK mutation was made in HBD domain.The mutant CPP possesses a good transduction activity and can bring fused EGFP protein into HeLa cells.