Rosenfeld S A, Ross O H, Hillman M C, Corman J I, Dowling R L
Dupont-Merck Pharmaceutical Company, Wilmington, Delaware 19880, USA.
Protein Expr Purif. 1996 Jun;7(4):423-30. doi: 10.1006/prep.1996.0063.
The cDNA that encodes the proenzyme form of human fibroblast collagenase (proMMP-1) was expressed in the methylotrophic yeast Pichia pastoris. The proMMP-1 encoding DNA was fused to the Saccharomyces cerevisiae pre-pro alpha-mating factor secretion signal in the P. pastoris pPIC9 expression plasmid, transformed into strain GS115 (His-), and His+ Muts (slow methanol utilization) transformants were selected. Full-length proenzyme and processed forms of the protein could be detected in yeast culture supernatants following shake flask and 10-liter fermentations. The protein was purified to greater than 95% homogeneity. The recombinant proMMP-1 was comparable to the native fibroblast material based on (i) migration of the full-length molecule as a 52-kDa protein on reducing SDS-PAGE, (ii) correct N-terminal amino acid sequence, (iii) activation of the full-length molecule by 4-amino-phenylmercuric acetate to yield processed protein species, (iv) degradation of gelatin as monitored by zymogram gels, and (v) enzymatic activity. These data suggest that the P. pastoris expression system offers a convenient and efficient means to produce and purify MMP-1.
编码人成纤维细胞胶原酶（proMMP-1）酶原形式的cDNA在甲基营养型酵母巴斯德毕赤酵母中表达。在巴斯德毕赤酵母pPIC9表达质粒中，将编码proMMP-1的DNA与酿酒酵母前原α-交配因子分泌信号融合，转化到GS115（His-）菌株中，并筛选His+ Muts（甲醇利用缓慢）转化子。在摇瓶培养和10升发酵后的酵母培养上清液中可检测到全长酶原和蛋白的加工形式。该蛋白被纯化至纯度大于95%。基于以下几点，重组proMMP-1与天然成纤维细胞物质相当：（i）在还原SDS-PAGE上全长分子作为52 kDa蛋白迁移；（ii）正确的N端氨基酸序列；（iii）用对氨基苯汞乙酸激活全长分子以产生加工后的蛋白种类；（iv）通过酶谱凝胶监测明胶的降解；（v）酶活性。这些数据表明，巴斯德毕赤酵母表达系统为生产和纯化MMP-1提供了一种方便且高效的方法。
