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Purification of alpha-hemolysin from an overproducing E. coli strain
Molecular & General Genetics : MGG
|January 1, 1985
Summary
Researchers cloned the alpha-hemolysin gene into E. coli, significantly increasing hemolysin production without harming the bacteria. This purified alpha-hemolysin is a 107 kDa polypeptide, challenging previous lethality assumptions.
Area of Science:
- Molecular Biology
- Microbiology
Background:
- Alpha-hemolysin is a toxin produced by certain E. coli strains.
- Previous assumptions suggested high hemolysin activity could be lethal to bacterial cells.
Purpose of the Study:
- To clone and express the alpha-hemolysin gene from plasmid pHly152.
- To investigate the production levels and cellular effects of high hemolysin activity.
- To purify and characterize the alpha-hemolysin protein.
Main Methods:
- Cloning the alpha-hemolysin determinant into pBR322, creating pSU157 and pSU158.
- Culturing E. coli strains with recombinant plasmids in minimal medium with hemoglobin.
- Purification of alpha-hemolysin using ammonium sulfate precipitation and gel filtration.
- Electrophoretic analysis of purified protein under denaturing conditions.
Main Results:
- Recombinant E. coli strains produced ~20 times more hemolysin activity than the parental strain.
- High hemolysin production was not lethal to the bacterial cells.
- Purified alpha-hemolysin appeared as a single 107 kDa polypeptide on denaturing gels.
- An Hly- mutant derivative did not synthesize this 107 kDa polypeptide.
Conclusions:
- The genetic determinant for alpha-hemolysin has been successfully cloned and expressed.
- High levels of alpha-hemolysin production are tolerated by E. coli.
- The alpha-hemolysin protein is a 107 kDa polypeptide.