尿激酶原在培养上清液中稳定性的研究

Stability of pro-urokinase in culture supernatant

目的:探讨尿激酶原糖基化对其表达稳定性的影响。方法:将302位天冬酰胺(A sn302)突变为丙氨酸(A la302),构建非糖基化尿激酶原。用基因重组的方法将重组尿激酶原和天然尿激酶原转染至dh-CHO细胞(二氢叶酸还原酶缺陷型——中国仓鼠卵巢细胞)中培养。收取无血清培养上清为检测标本。用嗜热杆菌的金属蛋白酶(ther-m o lys in)激活尿激酶原。用抑肽酶(aprotin in)终止激活反应。生色底物S-2444用于显色反应。在405nm波长处测定OD值(分光光度值)以确定双链成分的比例。在不用therm o lys in激活的条件下,用公式计算单链尿激酶原比例。将重组尿激酶原和天然尿激酶原的培养上清液置于4℃和37℃。每隔12 h测定总活性和单链成分比例。经过阳离子层析和凝胶层析纯化后,测定蛋白产品浓度、活性。以SDS-PAGE电泳鉴定蛋白质分子量和蛋白含量。结果:在37℃下天然尿激酸原(糖基化)的活性远高于重组尿激酶原(非糖基化)。无论4℃或37℃,糖基化尿激酶原的单链比例均高于非糖基化尿激酶原。纯化后的天然尿激酶原蛋白产品浓度和活性均高于重组尿激酶原。SDS-PAGE电泳结果提示天然尿激酶原浓度高于重组尿激酶原,分子量低于重组尿激酶原。结论:较高的温度加速原激酶原降解。A sn302位的糖基化位点有益于尿激酶原在培养上清液中的稳定性。

Objective： To investigate the effect of glycosylation on the stability of pro-urokinase expression. Methods： Non-glycosylated pro-urokinase was created by Asn302—Ala302 mutagenesis. The recombinant pro—UK and natural pro— UK were transflected into dhfr-CHO cells with gene recombinant technique. Serum-free culture supernatants were harvested for further assessment. Pro—UK was activated by thermolysin. The reaction was stopped by aprotinin. Chromogenic substrate S—2444 was used for chromogenesis. The form OD was measured at 405nm wavelength for detected amount of two-chain. Without thermolysin activation, single chain ratio was calculated with equation. The supernatants of recombinant pro—UK and natural pro—UK were stored at 4 ℃ and incubated at 37 ℃ separately. Total activity and single-chain ratio were detected every 12 hours. The concentrations and activities of pro— UK protein products were measured after ion-exchange chromatography and gel-filtration chromatography purifications. Protein concentration and molecular weight were assessed by SDS—PAGE. Results： The activity of natural pro —UK was much higher than that of non-glycosylated mutant at 37 ℃. The single-chain form of natural pro—UK was higher than those of non-glycosylated mutant at 4℃and 37 ℃. The protein concentration and activity of natural pro— UK were both higher than those of recombinant pro—UK. SDS—PAGE show that the concentration of natural pro—UK was higher than that of recombinant pro—UK and the molecular weight of natural pro—UK was lower than that of recombinant pro—UK. Conclusion： Higher temperature increases the proteolysis of pro—UK. The glycosylation site on Asn302 was benefit for pro—UK stability in culture supernatant.