Prokaryotic Expression and Purification of the Recombination Protein Containing Cell-penetrating TAT Peptide and Mouse SurvivinT34A Mutant: Investigation of Its Transduction Efficiency in Cells

Abstract: The expression and purification of the recombination protein TAT-msvT34A consisting of mice survivinT34A mutant fused with cell-penetrating TAT peptide and its transduction efficiency in several tumor and non-tumor cell lines were investigated. In this study， the prokaryotic expression vector pTAT-msvT34A was transformed into expression strain of E.coli BL21-DE3 and subsequently the transformed bacteria were induced by IPTG for expression of the recombinant protein. It was shown that the recombinant fusion protein was accumulated mainly in inclusion body and was purified up to a purity of 98% by the sequential application of affinity， ion exchange， and size-exclusion chromatographies. The transduction efficiency of the fusion protein labeled with FITC (FITC-TAT-msvT34A) in HepG2， TC-1， B16 and HEK293 cells was assayed by flow cytometry. It was shown that up to 60% of cells could be transduced at as low as the concentration 100 nmol/L of the protein. In contrast， the transduction efficiency of control BSA labeled with FITC was nearly neglectable， and it is statistically significant(P<0.01). On the other hand， the observation under fluorescence microscopy demonstrated that the transduction efficiency of TAT-msvT34A to HepG2 cell could reach up to 50% as well at the concentration 50 nmol/L and 100 nmol/L， while almost no transduction was observed in the cells treated with the control of FITC-labeled BSA. Therefore， we concluded that the prokaryotic expression fusion protein TAT-msvT34A could transduce HepG2， TC-1， B16 and HEK293 lines with high efficiency， which offered the potential for its application as an antitumor reagent in the future.

CSTR: 32200.14.cjcb.2011.07.0002