非特异性脂质转移蛋白突变体的构建及在两种硫氧还蛋白表达载体中的表达比较

Construction of the Mutants of Rice Nonspecific Lipid Transfer Protein and Expression Comparison in Two Kinds of Thioredoxin Fusion Expression Vectors

对水稻非特异性脂质转移蛋白 (Nospecificlipidtransferprotein ,nsLTP)LTP110中结构重要的 5个氨基酸位点进行了定点突变 ,测序结果证实了突变体构建成功。在尝试了多种大肠杆菌表达系统进行表达之后 ,发现硫氧还蛋白融合表达载体适合于LTP110野生型及突变体的表达。将编码野生型LTP110及突变体Y17A ,P72L ,R46A ,D45A ,C5 0A蛋白的cDNA顺序克隆进两种硫氧还蛋白表达载体并对其表达情况进行了比较 :pTrxFus载体可以在宿主菌GI72 4中以较低水平表达野生型LTP110及突变体Y17A ,P72L ,R46A融合蛋白 ,但不能表达D45A和C5 0A融合蛋白 ;pET32a(+)载体可以在宿主菌BL2 1(DE3)trxB- 中以可溶蛋白的形式表达野生型及所有突变型融合蛋白 ,且表达量比在pTrxFus载体 GI72 4突主菌中表达量高。对pET32a(+)载体中表达的LTP110融合蛋白进行了纯化 ,并利用带有荧光标记的脂肪酸分子对其测活 。

Five structural important residues of rice nonspecific lipid transfer protein LTP110 were mutated by site\|directed mutagenesis.Sequence results showed that they were all mutated successfully.After trying various \%E.coli\% expression systems,thioredoxin fusion expression system was found to be a proper system to express wild type and mutant LTP110.cDNA sequences ecoding wild type LTP110 and the mutants Y17A,P72L,R46A,D43A,C50A were cloned into two kinds of thioredoxin fusion expression vectors.The expression results were compared. In pTrxFus/GI724 expression system,wild type LTP110 and the mutants Y17A,P72L,R46A could be expressed at low level while D43A and C50A could not be expressed normally;in pET32a(+)/BL21(DE3) trxB - expression system,wild type LTP110 and all mutant proteins could be expressed very well and the levels were higher than that in pTrxFus/GI724 system.LTP110 fusion protein expressed in pET32a(+) vector was purified and its activity was checked by fluorescence labeled fatty acid.Results indicated that the recombinant LTP110 fusion protein has lipid binding activity.This work provides good basis for the further study.