尿激酶原糖基化对其表达稳定性的研究  

Effects of Glycosylation on the Stability of Pro-urokinase

作　　者：杨波[1] 李天德[1] 

机构地区：[1]中国人民解放军总医院心内科,北京100853

出　　处：《天津医药》2007年第3期211-213,I0003,共4页Tianjin Medical Journal

摘　　要：目的:探讨尿激酶原糖基化对其表达稳定性的影响。方法:构建非糖基化尿激酶原,收取无血清培养上清为检测标本。用嗜热杆菌金属蛋白酶(thermolysin)激活尿激酶原。用抑肽酶(aprotinin)终止激活反应。生色底物S-2444用于显色反应。在405nm波长处测定分光光度值(OD值)以确定双链成分的比例。在不用thermolysin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置于4℃和37℃。每隔12h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。SDS-PAGE电泳鉴定蛋白质分子质量和蛋白含量。结果:在37℃下天然尿激酶原(Asn302位带有糖基化侧链)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提示天然尿激酶原浓度高于重组尿激酶原,分子质量低于重组尿激酶原。结论:较高的温度加速尿激酶原降解。Asn302位的糖基化位点有益于尿激酶原在培养上清中的稳定性。Objective： To investigate the effects of glycosylation on the stability of pro-urokinase expression. Methods： Nonglycosylated pro-urokinase was constructed. Serum-free cultrue 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. OD value was measured at 405 nm wavelength in order to ensure the ration of two-chain form. Single chain ratio was calculated with equation without thermolysin activation. The supematants of recombinant pro-UK and natural pro-UK were stored at 4℃ and incubated at 37 ℃ separately. Total activity and single-chain ration were detected every 12 hours. The concentrations and activities of pro-UK protein products were measured after ion -exchange chromatography and gel-filtration chromatography pruifications. Protein concentration and molecaular weight were assessed by SDS-PAGE. Results： The activity of natural pro-UK was much higher than that of nonglycosylated mutant at 37℃. The single-chain form of natural pro-UK was higher than that of nonglycosylated 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 indicated 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 increased the proteolysis of pro-UK. The glycosylation site on Asn302 was benefit for pro-UK stability in culture supernatant.

关 键 词：尿纤溶酶原激活物 糖基化 嗜热菌蛋白酶 抑肽酶 基因表达 

分 类 号：R394.8[医药卫生—医学遗传学]