Prokaryotic Expression,Purification And Enzyme Activity Of Goldfish Tgf2 Transposase With Optimal Codons

Transposon is a kind of jumping gene that can change its insertion point in genome,and it can be diffused in natural different species through horizontal transfer.Active transposase,encoded by transposon,can starts transposition procedure in organism.However,many vertebrate transposons lost their activity because of mutation,and became non-autonomy activity transposons,so they could not complete its transposition procedure.In 2008,we found goldfish Tgf2 screened out from many goldfish strains,which was the second autonomy transposon after medaka Tol2,and belonged to hAT?hobo-AC-Tam?super family.Tgf2 transposon can code complete autonomy activity transposase,and has a wide application prospect in the field of fish transgentic.Our laboratory got 7 different Tgf2 transposon mRNA transcripts from goldfish embryo,and obtained 3 different animo acid lengths Tgf2 tranposases,686,650,and577,respectively.At present,these three lengths transposases were all expressed in E.coli.In order to further improve the expression and its expression stability,the 686transposase codons was optimized according to preference and degeneracy of the E.coli to codons.The purpose of the experiment is to express optimized 686 length Tgf2 transposase,and pET28a?+?,E.coli BL21?DE3?were used as plasmid vector and the expression host respectively.Meanwhile,we researched the method of keeping its activity stability.Four different probes that containing various repeat sequences were used to study Tgf2 transposase DNA binding activity and its specificity,and so to provide help for the construction of higher activity transposable enzymes.This experiment is divided into four parts.The first part is the construction of the recombinant expression vector.According to preference and degeneracy of the E.coli to codons,we optimized the goldfish Tgf2 transposase codons,named Tgf2-TPase⁸³.The cloning vector,Tgf2-TPase⁸³ gene sequence existed,and vector pET28a?+?was through double enzyme digestion using restriction endonucleases Bam I and XholI respectively,and then linked them together.We designed primers according to transposase and carrier to do PCR detect,identified by gene sequencing.Alignment result showed that the recombinant expression vector pET28a?+?-Tgf2-TPase⁸³ was constructed successfully.The second part is the prokaryotic expression,purification and identification of the recombinant vector pET28a?+?-Tgf2-TPase⁸³.In the experiment,we selected BL21?DE3?as the host bacteria,and the recombinant vector plasmid was expressed in it.The expression condition was:temperature was 30?,IPTG concentration was1.0 mM,induced time was 6 h,and the incubator speed was 150 rpm.Finally,we used SDS-PAGE?SDS polyacrylamide gel electrophoresis?to analyze.The result showed that at the molecular weight of about 79.6 kDa appeared protein band,and the protein band was deepened obviously after expression,so our target protein successfully expressed.In SDS-PAGE,we expressed soluble protein,so we adopted the methods of repeat freezing and thawing,ultrasonication,and centrifugal supernatant to purify the target protein through His Trap^TM HP column.Furthermore,we used different concentration lmidazole solution to eluent the target protein,and the purification sample was detected by SDS-PAGE,then the target protein band was cut and identified using mass spectrometry.The result showed that the expression amount up to 6.3%of the Tgf2-TPase⁸33 after optimization,and the purification was raised to 85%.Besides,the mass spectrometry result showed that the recombinant protein Tgf2-TPase⁸33 was the goldfish Tgf2 transposase.The third part is the preservation stability research of the recombinant protein Tgf2-TPase⁸³.In this study,we selected four different preservation methods:there were 20%glycerol-added-Tgf2-TPase⁸³,freezed directly-Tgf2-TPase⁸³,8%sucrose-added-Tgf2-TPase⁸³,and 4%mannitol-added-Tgf2-TPase⁸³,respectively,and they were preserved at-80?.Using plasmid pTgf2-EF1?-EGFP to detect its DNase activity after 7 d and 14 d,and the plasmid condition was detected through agarose gel electrophoresis.After scanning by BandScan,the change of the plasmid situation was quantified,so we obtained the various digestion speeds.The result showed that20%glycerol contributed to preserve Tgf2-TPase⁸33 enzyme activity,and other three preservation methods did not better than 20%glycerol.The fourth part is the research of the DNA binding activity and its specificity of the recombinant Tgf2-TPase⁸33 protein.Firstly,we decided wherther Tgf2-TPase⁸³transposase had DNA binding activity after preserved by 20%glycerol;secondly,goldfish Tgf2 transposon repeat sequence contains 5'AGTAA3'and 5'GTACT3',so four different repeat sequence amounts probes were designed:P_H,P_M1,P_M2,and P_L respectively.In experiment,glucosan gelchromatograph was used to decide whether Tgf2-TPase⁸33 had DNA binding activity,based on whether ultraviolet absorption value had an increase after protein and probes combination.Meanwhile,egg white lysozyme was called as negative control to decide whether the DNA binding activity had its specificity of the Tgf2-TPase⁸³.The result suggested that transposase had DNA binding activity with 20%glycerol added after 7 d,14 d and 30 d;different probes containing various repeat sequences had different DNA binding activity.The more the amount of the repeat sequences,the higher the binding activity.On the other hand,the lysozyme experiment showed that recombinant Tgf2-TPase⁸33 DNA binding activity was specific.The Tgf2 transposase,optimized through codons,not only increased its expression amount,but also improved its purification after purification.The soluble recombinant protein Tgf2-TPase⁸³ obtained from prokaryotic expression system not only provides a better effective method for fish transgenic,but also promotes transposon application in genetic transformation,fish breeding,and so on.