ABSTRACT
A general strategy for the expression of bacterial membrane transport and receptor genes in Escherichia coli is described. Expression is amplified so that the encoded proteins comprise 5-35% of E. coli inner membrane protein. Depending upon their topology, proteins are produced with RGSH6 or a Strep tag at the C-terminus. These enable purification in mg quantities for crystallization and NMR studies. Examples of one nutrient uptake and one multidrug extrusion protein from Helicobacter pylori are described. This strategy is successful for membrane proteins from H. pylori, E. coli, Enterococcus faecalis, Bacillus subtilis, Staphylococcus aureus, Microbacterium liquefaciens, Brucella abortus, Brucella melitensis, Campylobacter jejuni, Neisseria meningitides, Streptomyces coelicolor and Rhodobacter sphaeroides.
Subject(s)
Membrane Transport Proteins/metabolism , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Biological Transport, Active , Circular Dichroism , DNA, Bacterial/genetics , Helicobacter pylori/chemistry , Helicobacter pylori/genetics , Helicobacter pylori/metabolism , Membrane Transport Proteins/chemistry , Membrane Transport Proteins/genetics , Models, Biological , Plasmids , Restriction Mapping , Symporters/genetics , Symporters/metabolismABSTRACT
The biological roles of selenium and its mode of action have only recently begun to be revealed. To date, the major functions of selenium can be attributed to its antioxidative properties and its role in the regulation of thyroid hormone metabolism, cell growth and eicosanoid biosynthesis. The unusual feature of selenoprotein synthesis is that selenocysteine insertion is specified by the stop UGA codon. A number of selenocysteine-specific gene products and a stem-loop structure in the 3' untranslated region are required for selenocysteine biosynthesis and the decoding of UGA codons in the open reading frame of the mRNA. The major biological functions of selenium are achieved through its redox activity when present as selenocysteine at the active sites of selenoproteins and these proteins are selenium-dependent since replacement with the sulphur analogue cysteine causes loss of enzyme activity. Both organic and inorganic forms of selenium may be utilised by the body, with the selenoamino acids showing greatest bioavailability. Knowledge of the biochemistry of the element coupled with appropriate techniques for the study of the distribution of selenium species in health and disease could help to identify sensitive markers of selenium status.
