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Physical characterization of the cloned protease III gene from Escherichia coli K-12
Abstract:
Analysis of the cloned protease III gene (ptr) from Escherichia coli K-12 has demonstrated that in addition to the previously characterized 110,000-Mr protease III protein, a second 50,000-Mr polypeptide (p50) is derived from the amino-terminal end of the coding sequence. The p50 polypeptide is found predominantly in the periplasmic space along with protease III, but does not proteolytically degrade insulin, a substrate for protease III. p50 does not appear to originate from autolysis of the larger protein. Protease III is not essential for normal cell growth since deletion of the structural gene causes no observed alterations in the phenotypic properties of the bacteria. A 30-fold overproduction of protease III does not affect cell viability. A simple new purification method for protease III is described.
Insights
Researchers identified a new 50,000-Mr polypeptide (p50) from the protease III gene in Escherichia coli. This p50 protein is found in the periplasmic space but lacks protease III activity, and protease III is not essential for bacterial growth.
Area of Science:
- Molecular Biology
- Bacterial Physiology
Background:
- The protease III gene (ptr) in Escherichia coli encodes a 110,000-Mr protein.
- Protease III is located in the periplasmic space.
Purpose of the Study:
- To analyze the cloned protease III gene (ptr) from Escherichia coli K-12.
- To characterize a novel polypeptide derived from the protease III gene.
Main Methods:
- Gene cloning and analysis of Escherichia coli K-12.
- Protein characterization and localization studies.
- Enzyme activity assays using insulin as a substrate.
Main Results:
- A second polypeptide, p50 (50,000-Mr), is derived from the amino-terminal end of the protease III coding sequence.
- p50 is found in the periplasmic space with protease III but lacks proteolytic activity against insulin.
- Deletion of the protease III gene does not affect bacterial growth or viability, even with 30-fold overproduction.
Conclusions:
- The protease III gene produces at least two distinct polypeptides with differing functions.
- Protease III is not essential for normal Escherichia coli growth.
- A simplified purification method for protease III was developed.