Construction and characterization of Escherichia coli strains deficient in multiple secreted proteases: protease III degrades high-molecular-weight substrates in vivo.
Protease III, the product of the ptr gene, is a 110-kDa periplasmic protease with specificity towards insulin and other low-molecular-weight substrates (less than 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.
Baneyx
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Department of Chemical Engineering, University of Texas, Austin 78712.
Georgiou
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D015139:Blotting, Southern; D015153:Blotting, Western; D004247:DNA / Q000032:analysis; D010450:Endopeptidases / Q000502:physiology*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D007328:Insulin / Q000378:metabolism; D008666:Metalloendopeptidases; D010405:Penicillinase / Q000378:metabolism; D010957:Plasmids / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013205:Staphylococcal Protein A / Q000378:metabolism; D001618:beta-Lactamases / Q000378:metabolism
D007328:Insulin; D011993:Recombinant Fusion Proteins; D013205:Staphylococcal Protein A; D004247:DNA; D010450:Endopeptidases; D008666:Metalloendopeptidases; C046583:pitrilysin; D010405:Penicillinase; D001618:beta-Lactamases