Reilly T M, Seetharam R, Duke J L, Davis G L, Pierce S K, Walton H L, Kingsley D, Sisk W P
E. I. du Pont de Nemours and Company, Medical Products Department, Wilmington, Delaware 19880-0400.
J Biol Chem. 1990 Jun 5;265(16):9570-4.
A recombinant form of plasminogen activator inhibitor-1 (rPAI-1) has been purified from lysates of pCE1200, a bacterial expression vector containing the full length PAI-1 gene, by utilizing sequential anion exchange and cation exchange chromatography on Q-Sepharose and S-Sepharose columns. Approximately 140 mg of rPAI-1, estimated at 98% purity on the basis of analytical high performance liquid chromatography, could be obtained from 200 g wet weight of cells. The purified protein exhibited a single Coomassie Blue-stainable band at the region of Mr = 42,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and an NH2-terminal amino acid sequence consistent with the expected translation product of the pCE1200 PAI-1 insert. The rPAI-1 rapidly inhibited single- and two-chain tissue plasminogen activators, as well as urokinase, with apparent second order rate constants in the range of 2-5 x 10(7) M-1 s-1. A specific activity measurement of 250,000 units/mg was calculated for the rPAI-1 based on its ability to inhibit the enzymatic activity of a single-chain tissue plasminogen activator. Stability studies showed that the activity of the rPAI-1 was very stable when stored at temperatures of 25 degrees C or lower, but decayed within hours when stored at 37 degrees C. Sodium dodecyl sulfate treatment, which partially activates the latent form of natural PAI-1, inactivated rPAI-1. These results show that the purified rPAI-1 produced from pCE1200 displays many of the properties associated with the biologically active form of natural PAI-1.
一种重组形式的纤溶酶原激活物抑制剂-1（rPAI-1）已从pCE1200的裂解物中纯化出来，pCE1200是一种含有全长PAI-1基因的细菌表达载体，通过在Q-琼脂糖和S-琼脂糖柱上依次进行阴离子交换和阳离子交换色谱法。基于分析型高效液相色谱法估计纯度为98%，从200克湿重的细胞中可获得约140毫克的rPAI-1。通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳，纯化的蛋白质在Mr = 42,000区域呈现出一条考马斯亮蓝可染色带，其氨基末端氨基酸序列与pCE1200 PAI-1插入片段的预期翻译产物一致。rPAI-1能迅速抑制单链和双链组织纤溶酶原激活物以及尿激酶，其表观二级速率常数在2 - 5×10⁷ M⁻¹ s⁻¹范围内。基于其抑制单链组织纤溶酶原激活物酶活性的能力，计算出rPAI-1的比活性为250,000单位/毫克。稳定性研究表明，rPAI-1在25℃或更低温度下储存时活性非常稳定，但在37℃储存时几小时内就会衰减。十二烷基硫酸钠处理可部分激活天然PAI-1的潜伏形式，但会使rPAI-1失活。这些结果表明，从pCE1200产生的纯化rPAI-1具有许多与天然PAI-1生物活性形式相关的特性。
