重组玉米f型和m型硫氧还蛋白的功能确定及其靶向蛋白的捕获

Functional Determination of Recombinant Maize f and m Type Rhioredoxin and Entrapment of Target Proteins

RT-PCR从玉米幼叶总RNA中克隆f型和m型硫氧还蛋白(Thioredoxin,Trx)的编码基因,分别将两种类型Trx活性中心的第二个保守Cys残基定点突变成Ser残基和Ala残基。在大肠杆菌分别重组表达和纯化了含组氨酸标签的Trx及其突变体蛋白,SDS-PAGE显示纯化的蛋白显示一条主带,蛋白分子量分别估计为f型Trx为18kDa,m型Trx为14kDa;纯化的含有SUMO标签融合Trx,用SUMO专一性SUMO水解酶Ulp除去SUMO,等点聚焦电泳显示m型和f型Trx的等电点分别为4.6和5.9。m型Trx比f型Trx有更强的还原胰岛素能力,而突变体蛋白几乎没有还原能力。用Cys残基专一性标记化合物AMS标记Trx,显示野生型Trx有氧化还原态,而突变体蛋白仅有还原态。SDS-PAGE电泳显示固定化的f型Trx突变体比m型Trx突变体捕获的玉米幼叶靶蛋白更具有多样性。

The genes encoding the mature both types of thioredoxins,f and m type,were cloned by RT-PCR respectively using the total RNA from maize young leaves.The second conservative cystine residues in the catalytic site from two types of the proteins were mutated into serine and alanine residue respectively.The wild type and mutated thioredoxins with histidine-tag were overexpressed in Escherchia coli and purified.One band was displayed on SDS-PAGE.The molecular weight was estimated 18kDa for f type thioredoxin and 14kDa for m type thioredoxin.Both thioredoxins with SUMO fusion tags were also purified and the tags were removed with the SUMO protease Ulp.The proteins displayed pI values of 4.6 for Trx-m and 5.9 for Trx-f.The reduction of insulin suggested that the m type thioredoxin has more active than f type thioredoxin.Both mutated proteins hardly reduced insulin.The modification of the proteins by the specific cystine reagent AMS revealed that the purified wild type thioredoxins displayed the redox states,but the mutated thioredoxins showed the reduced state,suggesting that there is no disulfide bond in the mutated thioredoxins.The entrapped proteins from young leaves of maize by the mutated f type thioredoxin with histidine-tag immobilized on the Ni-NTA resin were more diverse than those by the mutated m type thioredoxin,as shown by SDS-PAGE.