关键词: 跨膜蛋白39A, 生物信息学分析, 融合蛋白, 条件优化, 可溶性表达

Abstract: Objective To construct prokaryotic expression vector of human TMEM39A, optimize expression conditions and obtain soluble TMEM39A. Methods TMEM39A gene was amplified from HEK293 cells total RNA by RT-PCR to construct its prokaryotic expression vector pGEX-6P-1-TMEM39A, and induced expression was conducted at different temperatures, IPTG concentrations, A600nm values and time conditions. Finally, expression of the recombinant protein GST-TMEM39A abundantly were carried out to analyze its solubility and antigenicity by using the best induction conditions. Results The nucleotide sequence homology of the recombinant expression vector pGEX-6P-1-TMEM39A and TMEM39A in GenBank (BC021277.2) was 99.9 % and the amino acid sequence homology was 100 %. The recombinant protein GST-TMEM39A showed two specific bands at 69 ku and 60 ku by Western blot analysis. The optimal induction conditions for GST-TMEM39A was 25 ℃, A600nm value of 0.6, 0.05 mmol/L IPTG induced 6 h. On this basis, the fusion expression of TMEM39A and GST protein was obtained in a soluble form. Conclusions The prokaryotic expression vector of TMEM39A is successfully constructed, the optimal expression condition of GST-TMEM39A is determined and its soluble expression is realized, which lay a material foundation for the subsequent purification of TMEM39A and the preparation of antibodies for functional studies, and provide scientific basis for further study about the relationship between TMEM39A and many diseases or viruses.

Key words: Transmembrane protein 39A (TMEM39A), Bioinformatics analysis, Fusion protein, Optimization of expression conditions, Soluble expression