还原SDS-PAGE电泳不适于鉴定尿激酶原单链比例

Reducing SDS-PAGE is not suitable for the detection of pro-urokinase single-chain form ratio

目的:探讨还原SDS-PAGE电泳检测尿激酶原单链比例的价值。方法:用PCR扩增的方法构建抗凝血酶的尿激原突变体。Arg精氨酸156-Lys赖氨酸156点突变可删除凝血酶作用位点。将突变体基因转染入dhfr-CHO细胞(dihydrofolate reductase deficient-Chinese hamster ovary cells二氢叶酸还原酶缺陷型—中国仓鼠卵巢细胞)。收取无血清培养上清液,用离子交换层析和凝胶过滤层析纯化抗凝血酶的尿激酶原(TRpro-UK)。结果:用还原SDS-PAGE电泳测得的尿激酶原单链比例是86.72%,而显色底物法为96.91%。而且随着电泳上样量的增加,单链比例明显下降。结论:还原SDS-PAGE电泳是在强还原条件下进行的,可能不宜用于鉴定尿激酶原单链比例。

Objective： To investigate the value of reducing SDS-PAGE to detect single-chain ratio of pro-urokinase （pro-UK）. Methods： A thrombin resistant pro-UK mutant （Arg156-Lys 156） was constructed with PCR amplification. The thrombin cleavage site was deleted by this mutagenesis. The mutant gene was transfected into dhfr- CHO cells （ dihydrofolate reductase deficient Chinese hamster ovary ceils）. Serum-free supernatant liquid was harvested and TRpro-UK （thrombin resistant pro-UK） purified with ion-exchange chromatography and gel filtration chromatography. Results： The single-chain ratio was 86.72M with reducing SDS-PAGE and 96.91% with chromogenic substrate assay, The single-chain ratio changed greatly with the variant sample concentrations. Conclusion： Reducing SDS-PAGE, which is under violate reducing condition, is not suitable for detecting pro-UK single-chain ratio.