CONSTRUCTION AND CHARACTERIZATION OF ESCHERICHIA-COLI STRAINS DEFICIENT IN MULTIPLE SECRETED PROTEASES - PROTEASE-III DEGRADES HIGH-MOLECULAR-WEIGHT SUBSTRATES INVIVO
CONSTRUCTION AND CHARACTERIZATION OF ESCHERICHIA-COLI STRAINS DEFICIENT IN MULTIPLE SECRETED PROTEASES - PROTEASE-III DEGRADES HIGH-MOLECULAR-WEIGHT SUBSTRATES INVIVO
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DOI：
10.1128/jb.173.8.2696-2703.1991
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发表时间：
1991-04-01
影响因子：
3.2
通讯作者：
GEORGIOU, G
中科院分区：
文献类型：
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作者：
BANEYX, F;GEORGIOU, G
蛋白酶III是ptr基因的产物，是一种110-kDa的周质蛋白酶，在体外对胰岛素和其他低分子量底物（<7，000分子量）具有特异性（Y. S.E. Cheng和D. Zipser，J.Biol.Chem.254：4698-4706，1979）。 通过插入失活ptr基因构建蛋白酶III缺陷的大肠杆菌菌株。 这种突变似乎没有影响相邻的recB和recC基因的功能。 蛋白A-β-内酰胺酶（一种蛋白酶敏感的分泌多肽）的表达在ptr细胞中增加约两倍。 通过脉冲追踪实验观察到蛋白A-β-内酰胺酶的半衰期相当地增加，这表明蛋白酶III参与体内高分子量底物的催化。除了生长速率的轻微降低之外，PTR突变体没有表现出可检测的表型改变。 当ptr突变转移到分泌型蛋白酶DegP缺陷的菌株中时，观察到生长速率进一步降低，以及融合蛋白表达的累加增加。 一个ptr degP ompT突变株导致在基本培养基中表达的进一步增加，但在丰富的培养基中没有。
Protease III, the product of the ptr gene, is a 110-kDa periplasmic protease with specificity towards insulin and other low-molecular-weight substrates (< 7,000 molecular weight) in vitro (Y.-S.E. Cheng and D. Zipser, J. Biol. Chem. 254:4698-4706, 1979). Escherichia coli strains deficient in protease III were constructed by insertional inactivation of the ptr gene. This mutation did not appear to affect the function of the adjoining recB and recC genes. Expression of protein A-beta-lactamase, a protease-sensitive secreted polypeptide, was increased approximately twofold in ptr cells. A comparable increase in the half-life of protein A-beta-lactamase was observed by pulse-chase experiments, suggesting that protease III is involved in the catabolism of high-molecular-weight substrates in vivo. ptr mutants exhibited no detectable phenotypic alterations except for a slight reduction in growth rate. When the ptr mutation was transferred to a strain deficient in the secreted protease DegP, a further decrease in growth rate, as well as an additive increase in the expression of the fusion protein, was observed. A ptr degP ompT mutant strain resulted in a further increase in expression in minimal medium but not in rich medium.