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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 coding genes of f-type and m-type thioredoxin (Trx) were cloned from total RNA of young leaves by RT-PCR. The second conserved Cys residues of the two types of Trx active sites were mutated to Ser Residues and Ala residues. The histidine-tagged Trx and its mutant proteins were recombinantly expressed and purified respectively in Escherichia coli. SDS-PAGE showed that the purified protein showed a main band with estimated protein molecular weight of 18 kDa for f-type and 14 kDa for m-type Trx ; SUMO-tagged fused Trx was purified, SUMO-specific SUMO hydrolase Ulp was used to remove SUMO, and isoelectric focusing was performed on the isoelectric point of type m and type f Trx by 4.6 and 5.9, respectively. The m-type Trx has a stronger ability to reduce insulin than the f-type Trx, while the mutant protein has almost no reducing power. Compound AMS labeled Trx with a Cys residue-specific marker shows that wild-type Trx has an oxidized and reduced form, whereas the mutant protein has only a reduced form. SDS-PAGE electrophoresis showed that the immobilized f-type Trx mutant was more diverse than the corn leaflet target protein captured by the m-type Trx mutant.