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尿激酶原， 即单链尿激酶（ｓｃｕＰＡ）是双链尿激酶（ｔｃｕＰＡ） 的前体， 属于丝氨酸蛋白酶家族， 但其内在酶活性高于一般的丝氨酸蛋白酶原。广泛存在于丝氨酸蛋白酶原家族中的ＡｓｐＨｉｓＳｅｒ 酶原三要素在尿激酶原中仅存Ａｓｐ， 为了恢复该结构， 降低尿激酶原的内在酶活性， 利用寡核苷酸介导的定点突变方法， 将尿激酶原基因中的特定核苷酸改变， 使Ａｌａ１７５ 突变为Ｓｅｒ１７５ ， 同时Ｔｙｒ１８７ 突变为Ｈｉｓ１８７ ， 得到尿激酶原变体（ｍｓｃｕＰＡ） 基因。该尿激酶原变体基因在大肠杆菌Ｅ．ｃｏｌｉ 中获得表达。经体外变、复性， 高流速ＳＰＳｅｐｈａｒｏｓｅ 柱层析，ＳｅｐｈａｃｒｙｌＳ２００ 凝胶层析， 表达产物被纯化。未突变的尿激酶原和尿激酶原变体对纤溶酶（ｐｌａｓｍｉｎ） 的敏感性表现一致。尿激酶原变体对人工合成的发色底物Ｓ２４４４（ｐｙｒｏＧｌｕＧｌｙＡｒｇｐｎｉｔｒｏａｎｉｌｉｎｅ）催化效率比尿激酶下降约２ ．５ 倍（ 表现为ｋｃａｔ／ Ｋｍ），对天然底物纤溶酶原（Ｇｌｕｐｌａｓｍｉｎｏｇｅｎ）的反应速度也有所下降。双链尿激酶和双链尿激酶变体（ｍｔｃｕＰＡ） 对Ｓ２４４４和天然底物纤溶酶原反应?
Urokinase, the single-chain urokinase (scuPA) is a precursor of double-chain urokinase (tcuPA), belongs to the serine protease family, but its intrinsic enzyme activity than the average serine protease. The Asp-His-Ser enzyme, which is widely present in the serine protease family, contains only Asp in pro-urokinase. To restore this structure and reduce the intrinsic enzymatic activity of pro-urokinase, oligonucleotide- Mutation method to change the specific nucleotide in the pro-urokinase gene to make Ala175 mutate to Ser175 and Tyr187 to His187 to obtain the urokinase variant (mscu-PA) gene. The urokinase variant gene is in E. coli E. coli. Obtain expression in E. coli. The extracellular, refolding, high-flow SP Sepharose column chromatography, Sephacryl S 200 gel chromatography, the expression product was purified. The unmutated urokinase and urokinase variants showed consistent sensitivity to plasmin. The pro-urokinase variant has a catalytic efficiency of about 2 less than that of urokinase on the synthetic chromogenic substrate S2444 (pyro-Glu-Gly-p-nitro-aniline). 5 times (expressed as kcat / Km), the reaction rate of natural substrate plasminogen (Gluplasminogen) also decreased. Double-chain urokinase and double-chain urokinase variant (mtcu PA) on the S2444 and natural substrate plasminogen reaction?