Construction of Prokaryotic Expression Vector,Expression,Purification,and Identification of Fusion Protein,TAT-RabGEF1
- VernacularTitle:融合蛋白TAT-RabGEF1原核表达载体的构建及蛋白表达
- Author:
Hong-Yi FANG
1
;
Na GUO
;
Ji-Bin XING
;
Chen-Fang LUO
Author Information
1. 中山大学附属第三医院麻醉科
- Keywords:
RabGEF1;
TAT protein;
fusion protein;
vector construction
- From:
Journal of Sun Yat-sen University(Medical Sciences)
2018;39(1):61-67
- CountryChina
- Language:Chinese
-
Abstract:
[Objective]To construct recombinant expression vector PET28a-TAT-RabGEF1,express,purify fusion pr-otein effectively in E.coli Rosetta(DE3),and investigate its transmembrane effect in vitro on P815 cells.[Methods]With cDNA in rats′tissues as the template,two primers containing the TAT sequence and two designed enzyme restriction cutting sites were designed. TAT-RabGEF1 fragment was amplified by PCR,and its product was inserted into PET-28a vector to construct recombinant plasmid PET28a-TAT-RabGEF1 prokaryotic expression vector. The recombinant vector was trans-formed into E.coli Rosetta(DE3)and express fusion proteins.The protein products were purified by affinity chromatography. The efficiency of the transduction of fusion protein into P815 cells were detected by immunofluorescence and analyzed by fluo-rescence spectro-photometer,with using methods of CCK-8 to analyze the cells viability after transduction of different con-centrations of fusion protein.[Results]The recombinant vector PET28a-TAT-RabGEF1 was constructed and the fusion pro-tein TAT-RabGEF1 was successfully expressed in E.coli Rosetta(DE3). By western blotting and SDS-PAGE we can see that the protein products′ relative molecular mass was about 57 ku,which was consistent with the target one,TAT-Rab-GEF1.The immunofluorescence results suggested that the fusion protein had the ability to transduct into P815 cells,and sat-uration of fusion proteins to be transducted into cells was 1 μmol/L.[Conclusion]Constructed recombinant vector PET28a-TAT-RabGEF1 and expressed fusion protein,TAT-RabGEF1,which verified the TAT′s ability of transduction. And it would build a solid technical foundation for the following research on the effect of RabGEF1′s on the activation of mast cells.
