|Title||Membrane protein expression in Lactococcus lactis.|
|Publication Type||Journal Article|
|Year of Publication||2015|
|Authors||King, MS, Boes, C, Kunji, ERS|
|Keywords||Cloning, Molecular, Gene Targeting, Genetic Vectors, Lactococcus lactis, Membrane Proteins, Transformation, Genetic|
The Gram-positive bacterium Lactococcus lactis has many properties that are ideal for the overproduction of membrane proteins in a functional form. Growth of lactococci is rapid, proceeds to high cell densities, and does not require aeration, which facilitates large-scale fermentation. The available promoter systems are strong and tightly regulated, allowing expression of toxic gene products in a controlled manner. Expressed membrane proteins are targeted exclusively to the cytoplasmic membrane, allowing the use of ionophores, ligands, and inhibitors to study activity of the membrane protein in whole cells. Constructed plasmids are stable and expression levels are highly reproducible. The relatively small genome size of the organism causes little redundancy, which facilitates complementation studies and allows for easier purification. The produced membrane proteins are often stable, as the organism has limited proteolytic capability, and they are readily solubilized from the membrane with mild detergents. Lactococci are multiple amino acid auxotrophs, allowing the incorporation of labels, such as selenomethionine. Among the few disadvantages are the low transformation frequency, AT-rich codon usage, and resistance to lysis by mechanical means, but these problems can be overcome fairly easily. We will describe in detail the protocols used to express membrane proteins in L. lactis, from cloning of the target gene to the isolation of membrane vesicles for the determination of expression levels.
|Alternate Journal||Meth. Enzymol.|
|Grant List||MC_U105663139 / / Medical Research Council / United Kingdom|