title: Genetic selection system for improving recombinant membrane protein expression in E. coli.
abstract: A major barrier to the physical characterization and structure determination of membrane proteins is low yield in recombinant expression. To address this problem, we have designed a selection strategy to isolate mutant strains of Escherichia coli that improve the expression of a targeted membrane protein. In this method, the coding sequence of the membrane protein of interest is fused to a C-terminal selectable marker, so that the production of the selectable marker and survival on selective media is linked to expression of the targeted membrane protein. Thus, mutant strains with improved expression properties can be directly selected. We also introduce a rapid method for curing isolated strains of the plasmids used during the selection process, in which the plasmids are removed by in vivo digestion with the homing endonuclease I-CreI. We tested this selection system on a rhomboid family protein from Mycobacterium tuberculosis (Rv1337) and were able to isolate mutants, which we call EXP strains, with up to 75-fold increased expression. The EXP strains also improve the expression of other membrane proteins that were not the target of selection, in one case roughly 90-fold.
authors.affiliation: Department of Chemistry and Biochemistry, University of California, Los Angeles, USA.
authors.forename: Elizabeth
authors.initials: E
authors.lastname: Massey-Gendel
authors.forename: Anni
authors.initials: A
authors.lastname: Zhao
authors.forename: Gabriella
authors.initials: G
authors.lastname: Boulting
authors.forename: Hye-Yeon
authors.initials: HY
authors.lastname: Kim
authors.forename: Michael A
authors.initials: MA
authors.lastname: Balamotis
authors.forename: Len M
authors.initials: LM
authors.lastname: Seligman
authors.forename: Robert K
authors.initials: RK
authors.lastname: Nakamoto
authors.forename: James U
authors.initials: JU
authors.lastname: Bowie
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D004262:DNA Restriction Enzymes / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism*; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D009169:Mycobacterium tuberculosis / Q000235:genetics; D010957:Plasmids / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D008565:Membrane Proteins; D011994:Recombinant Proteins; D004262:DNA Restriction Enzymes; C048901:endodeoxyribonuclease CreI