生物化学与生物物理进展
生物化學與生物物理進展
생물화학여생물물리진전
PROGRESS IN BIOCHEMISTRY AND BIOPHYSICS
2001年
2期
203-209
,共7页
党昕%杨晶鑫%茹强%茹炳根
黨昕%楊晶鑫%茹彊%茹炳根
당흔%양정흠%여강%여병근
PRGDWR片段%尿激酶原%溶栓活性%血小板聚集
PRGDWR片段%尿激酶原%溶栓活性%血小闆聚集
PRGDWR편단%뇨격매원%용전활성%혈소판취집
PRGDWR peptide%single chain urokinase-type plasminoge n activator%thrombolytic ability%platelet aggregation
为赋予单链尿激酶型纤溶酶原激活剂(single chain urokinase-type plasminogen activator, scu-PA, 尿激酶原)以抗血小板聚集的功能，在scu-PA的kringle区118 位Gly与119位Leu之间插入PRGDWR序列（insert mutant B，InB）.利用甲醇酵母（ Pichia pastoris）进行分泌表达，经金属离子螯合亲和层析与S强阳离子交换层析，得 到纯蛋白.实验测定InB对人工合成底物S-2444的酰胺解活性为5 900 IU/mg，动力学常数为 ：KInBm,S-2444=56.8 μmol*L-1，kInBca t, S-2444=0.33 s-1；水解天然底物plasminogen的动力学常数为：KInB m,plg=0.397 μmol*L-1，kInBcat,plg=0.0164 s-1 .InB激活plasminogen的反应在有fibrin存在条件下InB的活性为无fibrin条件下的46.3 ％.该突变体在体外激活plasminogen的活性与同一系统表达的野生型scu-PA基本相同.该突 变体表现出较强的抗血小板聚集活性，IC50＝12.7 μmol*L-1，而野 生型scu-PA无此功能.实验表明scu-PA的K区插入突变体InB是一种极具潜力的双功能溶栓 分子.
為賦予單鏈尿激酶型纖溶酶原激活劑(single chain urokinase-type plasminogen activator, scu-PA, 尿激酶原)以抗血小闆聚集的功能，在scu-PA的kringle區118 位Gly與119位Leu之間插入PRGDWR序列（insert mutant B，InB）.利用甲醇酵母（ Pichia pastoris）進行分泌錶達，經金屬離子螯閤親和層析與S彊暘離子交換層析，得 到純蛋白.實驗測定InB對人工閤成底物S-2444的酰胺解活性為5 900 IU/mg，動力學常數為 ：KInBm,S-2444=56.8 μmol*L-1，kInBca t, S-2444=0.33 s-1；水解天然底物plasminogen的動力學常數為：KInB m,plg=0.397 μmol*L-1，kInBcat,plg=0.0164 s-1 .InB激活plasminogen的反應在有fibrin存在條件下InB的活性為無fibrin條件下的46.3 ％.該突變體在體外激活plasminogen的活性與同一繫統錶達的野生型scu-PA基本相同.該突 變體錶現齣較彊的抗血小闆聚集活性，IC50＝12.7 μmol*L-1，而野 生型scu-PA無此功能.實驗錶明scu-PA的K區插入突變體InB是一種極具潛力的雙功能溶栓 分子.
위부여단련뇨격매형섬용매원격활제(single chain urokinase-type plasminogen activator, scu-PA, 뇨격매원)이항혈소판취집적공능，재scu-PA적kringle구118 위Gly여119위Leu지간삽입PRGDWR서렬（insert mutant B，InB）.이용갑순효모（ Pichia pastoris）진행분비표체，경금속리자오합친화층석여S강양리자교환층석，득 도순단백.실험측정InB대인공합성저물S-2444적선알해활성위5 900 IU/mg，동역학상수위 ：KInBm,S-2444=56.8 μmol*L-1，kInBca t, S-2444=0.33 s-1；수해천연저물plasminogen적동역학상수위：KInB m,plg=0.397 μmol*L-1，kInBcat,plg=0.0164 s-1 .InB격활plasminogen적반응재유fibrin존재조건하InB적활성위무fibrin조건하적46.3 ％.해돌변체재체외격활plasminogen적활성여동일계통표체적야생형scu-PA기본상동.해돌 변체표현출교강적항혈소판취집활성，IC50＝12.7 μmol*L-1，이야 생형scu-PA무차공능.실험표명scu-PA적K구삽입돌변체InB시일충겁구잠력적쌍공능용전 분자.
In order to obtain the bifunctional chimeric molecule of single-chain urokinase-type plasminogen activator (scu-PA) which can inhibit platelet aggregation, PRGDWR peptide was inserted into the site between Gly 118 and Leu119 (called insertion mutant B, InB). The recombinant gene of InB was expressed by Pichia pastoris. The secreted protein was purified by metal chelate affinity and strong cation exchange chromatography. The amidolytic ability of mutant InB is 5 900 IU/mg, the kinetic constants is: KInB m,plg=56.8 μmol*L-1，kInBcat,plg=0.33 s- 1. The kinetic constants of plasminogen activation reaction is: KInB m,plg=0.397 μmol*L-1，kInBcat,plg=0.0164 s-1. Fibrin inhibit the catalytiv ability of InB during plasminogen activatio n, the influence factor is 0.463(means InB remain 46.3% of the catalytic abili ty when fibrin was involved in the reaction system). The mutant not only has alm ost the same catalytic ability as wild type scu-PA, but also has strong ability of anti-platelet aggregation(compared with scu-PA), IC50 of InB is 12.7 μmol*L-1.
