高粱SbGABA-Ts基因的克隆、原核表达及纯化

Cloning,Prokaryotic Expression of Sorghum bicolor SbGABA-Ts

植物中GABA(γ-氨基丁酸)代谢与植物生长发育、信号传递及逆境响应等过程密切相关,而参与GABA代谢的关键酶GABA-T(γ-氨基丁酸转氨酶)在重要农艺作物中的研究相对滞后。利用同源性分析在高粱基因组数据库中获得两个γ-氨基丁酸转氨酶(SbGABA-T)基因,RT-PCR方法进行基因克隆,并连入原核表达载体pET28a(+),转化E.coli BL21(DE3)进行基因异源表达分析。结果表明,包含SbGABA-T编码区全长的融合蛋白主要在包涵体中表达,而去除了SbGABA-T N端信号肽的融合蛋白以部分可溶的形式存在。进一步优化表达条件,IPTG浓度为1 mmol/L时,16℃低温诱导18h,即可获得大量可溶性融合蛋白。用带His标签的镍柱对融合蛋白进行了纯化。

GABA is a ubiquitous four-carbon, non-protein amino acid, and has been associated with growth and development, signaling transduetion, and stress response in plants. GABA transaminase （ GABA-T ）, the enzyme responsible for the catabolism of GABA, has not been fully explored, especially in several important crops. Two putative GABA transaminase genes （GABA-T） from Sorghum bicolor were obtained based on homology analysis with the identified GABA-Ts of other plants and RT-PCR. Subsequently, the two SbGABA-T genes and corresponding N-terminal truncated SbGABA-Ts were inserted into pET28a （ ＋ ） vector and individually imported into E. coli strain BL21 （ pLysS, DE3 ） . Percentage improvement of soluble recombinant proteins were showed when removing the targeting peptide sequence of SbGABA-Ts. The fusion proteins were expressed partially in soluble form after incubating for 18 h at 16℃ by adding 1 mmol/L IPTG, and purified through nickel-affinity chromatography column.