Prokaryotic Expression,purification And In Vitro Activity Of Goldfish Tgf2 Transposase

Transposon is a kind of genetic factors that can move in the genome.The process is called transposition.With the development of the multiple genome sequencing project,transposons have been found in nearly all prokaryotes and eukaryotes.Transposons have been considered to be one of the important driving forces of variation and evolution in nature.Independent DNA transposons can encode transposase to catalyze transposition process.However,because of vertical inactivation from the host,few transposons have active.The Tgf2 transposon was the second vertebrate transposons which has natural activity.The Tgf2 belongs to the h AT superfamily which was first found in the goldfish(Carassius auratus).The transposon can encode integrated and activated transposase which can mediate transposition.Therefore,the Tgf2 transposon has great potential in applications such as screening master genes of important traits,functional explanation of genes and fish breeding.The Tgf2 transposon is able to encode three Tgf2 transposases in this study.At present,the three different lengths of Tgf2 transposase are expressed in E.coli Rosetta(DE3).Exogenous proteins is expressed by E.coli BL21(DE3)strain with higher effic iency and stability,so Tgf2 transposase(Tgf2TP)was optimized based on codon preference of E.coli.The synthesized Tgf2 TP Plus was then cloned into p ET-28a(+)and transformed into BL21(DE3)cells to obtain high expression of Tgf2 transposase.This study is divided into five portions.The component of the first part is bioinformatics analys is of Tgf2 transposase.The secondary structure,domain and tertiary structure of transposase were analyzed by SOPMA and I-TASSER servers.The tertiary structure of the transposase was simulated;the catalytic element of the transposase was found,and the functional domain associated with the transposase activity was analyzed.The component of the second part is the construction of prokaryotic expression plasmid of the goldfish Tgf2 transposase.For existence of many rare codons in Tgf2 transposase c DNA,Tgf2 TP Plus was optimized based on codon preference of E.coli.The cloning vector p UC57-simple-Tgf2 TP Plus with Tgf2 transposase gene sequences and expression vector p ET-28a(+)were incised by restriction enzyme Bam HI and XhoI.Then the fragments of Tgf2 TP Plus and p ET-28a(+)were purified from agarose gel and ligated using T4 DNA ligase.The recombinant vector transformed into E.coli DH5? cells.The suitable primers were designed according to the recombinant vector sequences.The recombinant vector identified by PCR detection and gene sequencing method.The results showed that the prokaryotic expression vector p ET-28a(+)-Tgf2 TP Plus was constructed successfully.The component of the third part is the expression of recombinant protein.The recombinant plasmid p ET-28a(+)-Tgf2 TP Plus was extracted from E.coli DH5? cells and transformed into E.coli BL21(DE3)cells.The recombinant protein was induced by IPTG induction.The results showed that the recombinant protein was no expression at low absorbance(OD600=0.3-0.4)and low temperature(22 ?),but it was expressed at 37 ? and OD600=0.5.Then time,concentration of IPTG and temperature were optimized.The results showed that the expression quantity of the recombinant protein is increase gradually with the extension of induction time(0-6 h),but the expression quantity of the recombinant protein is no change with the extension of induction time(6-10 h).And the concentration of bacteria is higher over 6 h.Therefore,the induction time was 6h.When the concentration of the induction was 1.0 mmol/L,the expression quantity of the target protein was the highest.In this study,the concentration of IPTG was 1.0 mmol/L.From 28 ? to 37 ?,the expression of recombinant protein was higher at 28 ? and 30 ?,And the concentration of bacteria is higher at 30 ?.So this experiment was taken at 30 ? to induce expression.The study chose that the recombinant protein was induced in OD600=0.5,1.0 mM IPTG for 6 h.SDS-PAGE analys is showed significant protein bands at ~70 kDa,consistent with predicted molecular weight of Tgf2 transposase.The component of the fourth part is the purification and identification of the recombinant proteins.The E.coli BL21(DE3)strain was able to express soluble recombinant proteins.The recombinant protein carries the His?Tag,so,the recombinant protein was purified by Ni2+ column affinity chromatography.Then,collecting bacterias,the ultrasonic crusher breaks the cell.The supernatant was loaded onto a Ni+2-affinity column.The target fractions were analyzed by SDS-PAGE.The results showed that the recombinant protein was obtained with high purity,and the molecular weight of the recombinant protein was consistent with the predicted molecular weight of Tgf2 transposase.The recombinant protein was identified as Tgf2 transposase of goldfish by MALDI-(TOF)/ TOF.The component of the fifth part is the activity in vitro of Tgf2 transposase.Activities in vitro include DNA binding activity and digestive activity.The DNA binding activity experiment was researched by dextran gel chromatography.The DNA binding activity is judged by the changes of the UV absorbance.The transposase and plasmid were incubated.Agarose gel electrophoresis was used to detect the mixture.The DNA probe is the terminal repeat sequence of the left arm of the Tgf2 transposon.The results showed that goldfish Tgf2 transposase can bind to the probe specifically,and the binding activity is not affected by the concentration of probe.Tgf2 transposase has digestive activity.The prokaryotic expression system for Tgf2 transposase not only provides soluble and active transposase efficiently,but also establishes foundation for Tgf2 TP Plus used as enzyme tools in a variety of fish biology research and construct transposase with higher activity.