Prokaryotic expression of goldfish Tgf2 transposase with optimal codons and its enzyme activity  

作　　者：Xi Zhao Ruirui Si Mingjuan He Xiayun Jiang Shuming Zou 

机构地区：[1]College of Food Science and Technology,Shanghai Ocean University,Shanghai,201306,China [2]Genetics and Breeding Center for Blunt Snout Bream,Key Laboratory of Freshwater Aquatic Genetic Resources,National Demonstration Center for Experimental Fisheries Science Education,Shanghai Ocean University,Shanghai,201306,China

出　　处：《Aquaculture and Fisheries》2019年第3期98-104,共7页渔业学报（英文）

基　　金：This work was supported by the National Science Foundation of China(31572220,31272633,31201760);the National High Technology Research and Development Program of China(863 Program)(2011AA100403);the Shanghai University Knowledge Service Platform(ZF1206).

摘　　要：Tgf2 transposase(Tgf2-TPase),a hAT transposase from goldfish,plays an important role in fish transgenic applications.Previously,the production of the recombinant Tgf2-TPase protein required rigorous fermentation at low temperatures(22℃)and early log phase induction(OD600=0.3–0.4)in Rosetta 1(DE3)Escherichia coli lines.In order to better express the Tgf2-TPase and detect its enzyme activity,83 rare codons in Tgf2-TPase were optimized and designated Tgf2-TPase^(83).The expression results showed that the soluble recombinant Tgf2-TPase83 was highly expressed at 30℃ and was inducible at an OD600 of 0.5–0.6 in the same prokaryotic expression system.After purification by affinity chromatography,Tgf2-TPase83 with codon optimization had higher enzyme activity than the Tgf2-TPase control.Comparison of different preservation methods(freezedrying at−80℃,storage in 20%-glycerol,8%-sucrose,4%-mannitol),revealed storage of Tgf2-TPase^(83) in glycerol helped to preserve its DNase digestion activity.Furthermore,size exclusion chromatography suggested that the purified Tgf2-TPase^(83) could recognize and bind to DNA probes containing a terminal inverted repeat(TIR)and a subterminal repeat(STR)sequence of the Tgf2 transposon.Overall,the results showed that optimizing the 83 codons of Tgf2 transposase can simplify the fermentation process and improve the enzyme activity.We propose that the production of the Tgf2-Tpase83 protein in a soluble and active form could provide an alternative tool for genetic modification of fish.

关 键 词：Codon optimization Enzyme activity Prokaryotic expression Tgf2 transposase 

分 类 号：Q78[生物学—分子生物学]