KefC is a potassium transport system of <i>E.coli</i> that is regulated by glutathione metabolites. An analysis of the KefC protein was undertaken in order to advance towards an understanding of the transport and regulatory processes at a molecular level. KefC-LacZ hybrid proteins were constructed by mini-Mu transposon insertion mutagenesis into <i>kefC</i> plasmids. The distribution of the B-galactosidase activity between membrane and soluble fractions indicated that the KefC moieties of the hybrid proteins were directing the proteins to the membrane, suggesting that KefC is a membrane protein. Sequencing the fusion junctions of the <i>kefC'-'lacZ</i> gene fusions allowed progress to be made towards topological mapping of the KefC protein. Two stable, high activity hybrid proteins confirmed the location of the first cytoplasmic loop and the large, cytoplasmic C-terminal domain proposed on the basis of the deduced amino acid sequence. A knowledge of the gene orientation, derived from restriction mapping of the transposon insertions, enabled kefC to be cloned downstream of a bacteriophage T7 promoter and expressed using the T7 polymerase/promoter system, overcoming initial problems of low expression. This identified KefC as a membrane located protein of apparent molecular mass 55-60 kDa. The oligomerization of KefC was investigated. It was shown that treatment with the cross-linking reagent formaldehyde moved the KefC band to a higher molecular weight and it was suggested that KefC-LacZ hybrid proteins interfered with potassium efflux via KefC. These observations are consistent with KefC functioning as an oligomer. Regimes suitable for solubilization and purification of KefC-LacZ hybrid proteins were developed. Attempts were made to generate antibodies against KefC but encountered difficulties due to contamination with antibodies not specific for KefC.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:290398 |
| Date | January 1990 |
| Creators | Ritchie, Graeme Y. |
| Publisher | University of Aberdeen |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
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