Construction of TAT-KLF4 vector and expression of fusion protein

Abstract

Objective To construct recombinant expression vector TAT-KLF4 and express the Fusion Protein in E. coli BL21. Methods The open reading frame(ORF) of KLF4 gene was amplified by PCR. The PCR product was digested with restriction enzymes then was inserted into PET-28b-TAT-V2 which contained protein transduct domain of TAT. The recombinant vector was transfected into E. coli BL21 and the expression of the TAT-KLF4 fusion protein was induced with IPTG. The fusion protein was analyzed by using SDS- PAGE and purified by the affinity chromatography. Western Blot was used to identify the specificity of the fusion protein. The immunofluorescence was used to detection the efficiency of fusion protein transducted to HSF cells. Results The recombinant expression vector TAT-KLF4 was correctly constructed and fusion Proteion TAT-KLF4 was successfully expressed in prokaryotic cells. Western blotting showed the specificity of the fusion protein. The immunofluorescence prompt fusion protein transduced into HSF cells quickly. Conclusion This study provides the material basis for further induction of the induced pluripotent stem cells by protein transduction. Key words: Protein KLF4; Stem cells; Protein structure, quaternary; Genetic vectors