哈茨木霉丝/苏氨酸蛋白磷酸酶基因的克隆表达

为深入研究丝/苏氨酸蛋白磷酸酶的功能,从哈茨木霉cDNA文库中克隆丝/苏氨酸蛋白磷酸酶(PP2A)基因,并成功构建到原核表达载体pET28a,在大肠杆菌中表达.表达产物经SDS-PAGE电泳分析,在40.5kDa处出现特异性条带.在22℃以0.5mmol/L IPTG诱导时,目的蛋白大部分以可溶形式存在,在37℃以1.0mmol/L IPTG诱导时,目的蛋白则大部分以包涵体存在.全长cDNA序列为1286bp,5′非编码区91bp、3′非编码区237bp,编码327个氨基酸,没有信号肽.本研究为PP2A基因的功能验证奠定基础,以便进一步研究蛋白磷酸酶的功能,从而获得更多关于哈茨木霉蛋白磷酸酶作用机制的信息.

Gene cloning, prokaryotic expression of PP2A gene from Trichoderma harzianum

In order to investigate the function of serine/threonine protein phosphatase (PP), the full length cDNA of PP2A was obtained from cDNA library of Trichoderma harzianum and the recombinant prokaryotic expression vector pET28a was constructed. The vector was transformed into E. coli BL21 (DE3). The expressed fusion protein was analyzed by SDS-PAGE, and a new specific band with molecular weight of about 40.5 kDa was found when induced by IPTG. The target protein was dissoluble when induced at 22 ℃ with IPTG of 0. 5 mmol/L, while insoluble if induced at 37 ℃ with IPTG of 1.0 mmol/L. The entire cDNA sequence consisted of 1286 bp with 91 and 237 bp in 5′ and 3′ untranslated regions respectively. The gene encoding 327 amino acids had no signal peptide sequence. These results can provide a basis for validating the functions of PP2A. The structure and function of PP2A can be further studied and more information of phosphorylation mechanism in T. harzianum can be obtained.