Steven J, Cottingham I R, Berry S J, Chinery S A, Goodey A R, Courtney M, Ballance D J
Delta Biotechnology Ltd, Nottingham, England.
Eur J Biochem. 1991 Mar 14;196(2):431-8. doi: 10.1111/j.1432-1033.1991.tb15834.x.
Expression of plasminogen activator inhibitor 2 (PAI-2) under the control of the protease B gene promoter in a mutant strain of Saccharomyces cerevisiae, DS569, resulted in its accumulation intracellularly at up to 20% of the soluble cell protein. Provision of an N-terminal signal sequence resulted in the secretion of a hyperglycosylated molecule. The intracellularly produced PAI-2 was purified by copper-chelate and anion-exchange chromatography to greater than 95% pure and was fully active. The recombinant PAI-2 formed SDS-stable complexes with urokinase and tissue-type plasminogen activator and inhibited the proteases with similar reaction kinetics to placental PAI-2 (second-order rate constant for uPA, 2.4 x 10(6) M-1 s-1, and for two-chain tPA, 0.7 x 10(5) M-1 s-1). As is the case for placental PAI-2, the N-terminus of the yeast-derived recombinant PAI-2 was blocked. The high productivity and consequent ease of purification mean that S. cerevisiae provides an excellent source of recombinant PAI-2 for investigation of its therapeutic potential in the treatment of neoplastic and inflammatory diseases.
在酿酒酵母突变株DS569中，在蛋白酶B基因启动子的控制下表达纤溶酶原激活物抑制剂2（PAI-2），导致其在细胞内积累，含量高达可溶性细胞蛋白的20%。提供N端信号序列导致分泌一种高度糖基化的分子。通过铜螯合和阴离子交换色谱法纯化细胞内产生的PAI-2，纯度大于95%，且具有完全活性。重组PAI-2与尿激酶和组织型纤溶酶原激活物形成SDS稳定的复合物，并以与胎盘PAI-2相似的反应动力学抑制蛋白酶（尿激酶的二级速率常数为2.4×10⁶ M⁻¹ s⁻¹，双链组织型纤溶酶原激活物的二级速率常数为0.7×10⁵ M⁻¹ s⁻¹）。与胎盘PAI-2一样，酵母来源的重组PAI-2的N端被封闭。高产量以及随之而来的易于纯化意味着酿酒酵母为研究其在治疗肿瘤和炎症性疾病中的治疗潜力提供了一种极好的重组PAI-2来源。
