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Amino-terminal sequence analysis of four plasmid-encoded virulence-associated proteins of Salmonella typhimurium
S Taira1, M Baumann, P Riikonen
1National Public Health Institute, Molecular Biology Unit, Helsinki, Finland.
Abstract:
We have expressed four virulence-associated proteins encoded by the Salmonella typhimurium plasmid pEX102 in Escherichia coli. The genes coding for the proteins MkaA, MkaB, MkaC and MkaD subcloned in the vectors pUC19 or Bluescript KS+ directed substantial production of these proteins in E. coli. The same host harbouring pEX102 did not produce these proteins in detectable amounts. The proteins were exclusively found in the particulate fraction. The amino-terminal sequence analysis showed that the N-termini of these proteins corresponded to the ones predicted from the open reading frames found previously in the DNA sequence of the virulence determinant and that no N-terminal signal sequence processing of the proteins occurred.
Insights
Researchers successfully expressed four Salmonella typhimurium virulence proteins (MkaA, MkaB, MkaC, MkaD) in Escherichia coli. These proteins were found in the particulate fraction, with no signal sequence processing observed.
Area of Science:
- Microbiology
- Molecular Biology
- Bacteriology
Background:
- Salmonella typhimurium is a significant pathogen.
- Virulence factors contribute to bacterial pathogenicity.
- Plasmid-encoded genes often play a role in virulence.
Purpose of the Study:
- To express and characterize four virulence-associated proteins (MkaA, MkaB, MkaC, MkaD) from Salmonella typhimurium.
- To investigate the production and localization of these proteins in a heterologous host.
Main Methods:
- Gene cloning and subcloning of MkaA, MkaB, MkaC, and MkaD into expression vectors (pUC19, Bluescript KS+).
- Heterologous protein expression in Escherichia coli.
- Fractionation of bacterial cell components.
- Amino-terminal sequence analysis.
Main Results:
- Substantial production of MkaA, MkaB, MkaC, and MkaD was achieved in E. coli.
- These proteins were not detected when expressed from the native plasmid (pEX102) in E. coli.
- The expressed proteins were localized to the particulate fraction of the E. coli cells.
- Amino-terminal sequencing confirmed the predicted protein sequences and indicated no signal peptide cleavage.
Conclusions:
- Heterologous expression systems can be used to produce Salmonella virulence proteins.
- The expression of these proteins from the native plasmid in E. coli is inefficient or regulated.
- The Mka proteins are likely membrane-associated or intracellular particulate components.