Prokaryotic Expression of piggyBac Transposase with N-terminal Fused Human immunodeficiency virus (HIV) Transactivator (TAT) Peptide and Verification of Its Activity in HEK 293 Cells
Abstract：To enhance the safety of piggyBac (PB) transposon system and weaken the genotoxicity of the helper plasmid in gene transfer, we try to use PBase N-terminal fused Human immunodeficiency virus (HIV)transactivator (TAT) peptide with prokaryotic expression to replace the helper plasmid and verify its activity in human embryonic kidney 293 cells (HEK 293 cells). Furthermore, we hope to establish an approach that TAT peptide mediates foreign proteins into eukaryotic cells. In present study, we constructed two prokaryotic expression vectors containing the plasmid pET28A-Pbase 1 to generate PBase N-terminal fused TAT peptide and the plasmid pET28A-Pbase 2 to generate PBase C-terminal fused TAT peptide. The PBase with N-terminal fused TAT peptide was successfully expressed by the plasmid pET28A-Pbase 1 in Escherichia coli BL21 (DE3) strain. Polyacrylamide gel electrophoresis (PAGE) and Western blot (WB) were used to confirm the PBase expression. After protein quantification, the PBase with 100 mg/mL in cell culture medium was transduced with the donor plasmid into HEK 293 cells. Furthermore, HEK 293 cells transfected the dual plasmid system of PB transposon were the positive control. PBase were determined by immunofluorescent staining (IF) in HEK 293 cells. Thermal asymmetric interlaced PCR (Tail-PCR) was used to detect the flanking sequences of PB integrate sites from purified genome of HEK 293 cell clones. After methylene blue staining for HEK 293 cell clones, Student’s t-test was employed to analyze the colony count. Results showed that the PBase was successfully expressed by the form of soluble protein. Simultaneously, we found that the PBase was localized in the nucleus from the result of immunofluorescent staining. Detection of flanking sequences indicated that the integration mechanism of PB was regarded as random recombination. Compared the dual plasmid system of PB, HEK 293 cell clones generated by the PBase with prokaryotic expression were significantly reduced and similar to negative control. These observations suggested that the PBase with prokaryotic expression lost its natural activity and caused the failure of transposition. Hence, further studies are necessary for this PB transposon system lead to transposition in other eukaryotic cells. However, the comprehensive information from this study supplied a reliable method for foreign proteins mediated with TAT peptide across eukaryocyte membranes.
胡广东1,高元鹏2,王静2,郑丽明2,张涌1. N端融合TAT穿膜肽的piggyBac转座酶的原核表达及其在HEK 293细胞中的功能验证[J]. , 2014, 22(4): 406-414.
1, 1, 1, 1,. Prokaryotic Expression of piggyBac Transposase with N-terminal Fused Human immunodeficiency virus (HIV) Transactivator (TAT) Peptide and Verification of Its Activity in HEK 293 Cells. , 2014, 22(4): 406-414.
