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Title: Lactococcus lactis : an alternative expression system for the over-production and functional characterisation of mitochondrial carriers
Author: Chan, K. W.
Awarding Body: University of Cambridge
Current Institution: University of Cambridge
Date of Award: 2006
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A family of membrane proteins, known as mitochondrial carriers, transport metabolites, cofactors and nucleotides across the inner membrane of mitochondria. When the carriers are expressed in Escherichia coli, inclusion bodies are formed, which contain misfolded and dysfunctional material. Mitochondrial carriers can be functionally over-expressed in yeast mitochondria, but require purification for functional studies. Here, the nisin expression system in the Gram-positive bacterium Lactococcus lactis was used for the expression of mitochondrial carriers. The results show that the carriers are over-produced in the cytoplasmic membrane and are functional. The expression studies have revealed possible complications during insertion, because the amino-terminal region has to be transported to the outside of the cell. These limitations were overcome by the exchange and truncation of the T-terminal region or by the addition of the N-terminal lactococcal signal peptides for correct targeting and insertion of the carriers. The expression system was used for the functional identification of a single mitochondrial carrier from the human parasitic amoeba Entamoeba histolytica. This parasite lacks typical mitochondria, but may has a highly reduced mitochondrai remnant called the mitosome. The Entamoeba carrier was expressed as a N-terminal truncation mutant transporting ATP and ADP. A novel mechanism not reliant on a membrane potential was used, making it distinct from archetypal mitochondrial ADP/ATP carriers. In addition, we have successfully demonstrated purification and functional reconstitution of the yeast mitochondrial GTP/GDP carrier after it was over-expressed in lactococcal membranes. In conclusion, the lactococcal expression system may be a promising way to identify and characterise mitochondrial carriers or other eukaryotic membrane proteins.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available