金鱼Tgf2转座酶的原核表达及其DNA结合活性

金鱼Tgf2转座子基因属于Hobo/Activator/Tam3(hAT)超家族,其编码的活性转座酶能在多种鱼类转座中介导基因插入及诱变,因此在鱼类重要性状主控基因的筛选、功能解释及育种等方面具有重要的应用前景.鉴于Tgf2转座酶基因(JN886591)存在众多稀有密码子,本研究依据大肠杆菌对密码子的偏好性,对金鱼Tgf2转座酶基因进行密码子优化,所得Tgf2转座酶基因连接原核表达载体pET-28a(+),将重组载体转化到大肠杆菌BL21 (DE3)细胞.该大肠杆菌细胞在培养温度37℃、OD600≈0.5时用IPTG诱导获得高效表达,即菌体中含有8.8％的重组蛋白,上清液中含有4.0％重组蛋白.采用亲和层析纯化从菌体上清液中分离得到分子量约70 ku的可溶性重组蛋白,经MALDI-(TOF)/TOF串联质谱鉴定其为重组Tgf2转座酶.进而采用分子排阻色谱法研究转座酶的DNA结合活性,结果表明:所得重组Tgf2转座酶能识别并结合含Tgf2转座子特异性亚末端重复序列的DNA探针,即启动转座.采用原核表达体系高效获得重组Tgf2转座酶,为其作为工具酶应用于鱼类生物学研究奠定必要的基础.

Prokaryotic expression of goldfish Tgf2 transposase and its DNA binding activity

Goldfish Tgf2,a transposon of Hobo/Activator/Tam3 (hAT) superfamily,encodes active transposase which is capable of mediating gene insertion and mutagenesis in a series of fish species.Therefore,it has great potential in applications such as screening master genes of important traits,functional explanation of genes and fish breeding.For existence of many rare codons in Tgf2 transposase cDNA (Tgf2TP,JN886591),Tgf2TP was optimized based on codon preference of E.coli.The synthesized Tgf2TP was then cloned into pET-28a(+) and transformed into BL21 (DE3) cells.After incubation at temperature (37 ℃) and induction when OD600 ≈0.5,the cells produced high yield of recombinant proteins,which accounted for 8.8％ of total proteins in cells and 4.0％ of supernatant proteins in lysates.By affinity chromatography purified recombinant protein (70 ku) was obtained,subjected to MALDI-(TOF)/TOF and identified as Tgf2 transposase (Tgf2TP).Then size exclusion chromatography was applied to evaluate DNA-binding activity of Tgf2TP.The results showed that recombinant Tgf2TP recognized and bound specific DNA probes containing sub-terminal repeat sequences of Tgf2,which means start of transposition.The prokaryotic expression system for Tgf2TP not only provided soluble and active transposase efficiently,but also laid a foundation for Tgf2TP used as enzyme tools in a variety of fish biology research.