title: Topology-informed strategies for the overexpression and purification of membrane proteins.
abstract: Membrane proteins represent a significant fraction of all genomes and play key roles in many aspects of biology, but their structural analysis has been hampered by difficulties in large-scale production and crystallisation. To overcome the first of these hurdles, we present here a systematic approach for expression and affinity-tagging which takes into account transmembrane topology. Using a set of bacterial transporters with known topologies, we tested the efficacy of a panel of conventional and Gateway recombinational cloning vectors designed for protein expression under the control of the tac promoter, and for the addition of differing N- and C-terminal affinity tags. For transporters in which both termini are cytoplasmic, C-terminal oligohistidine tagging by recombinational cloning typically yielded functional protein at levels equivalent to or greater than those achieved by conventional cloning. In contrast, it was not effective for examples of the substantial minority of proteins that have one or both termini located on the periplasmic side of the membrane, possibly because of impairment of membrane insertion by the tag and/or att-site-encoded sequences. However, fusion either of an oligohistidine tag to cytoplasmic (but not periplasmic) termini, or of a Strep-tag II peptide to periplasmic termini using conventional cloning vectors did not interfere with membrane insertion, enabling high-level expression of such proteins. In conjunction with use of a C-terminal Lumio fluorescence tag, which we found to be compatible with both periplasmic and cytoplasmic locations, these findings offer a system for strategic planning of construct design for high throughput expression of membrane proteins for structural genomics projects.
authors: Astbury Centre for Structural Molecular Biology, Leeds, UK.
authors: Moazur
authors: M
authors: Rahman
authors: Fouzia
authors: F
authors: Ismat
authors: Michael J J
authors: MJ
authors: McPherson
authors: Stephen A
authors: SA
authors: Baldwin
mesh_terms: D015153:Blotting, Western; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism; D005822:Genetic Vectors / Q000235:genetics; D008565:Membrane Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D019897:Periplasm / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D027981:Symporters / Q000235:genetics / Q000378:metabolism
chemical_list: D029968:Escherichia coli Proteins; D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins; D027981:Symporters; C073766:GltP protein, E coli