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Genetic selection system for improving recombinant membrane protein expression in E. coli
Author(s) -
MasseyGendel Elizabeth,
Zhao Anni,
Boulting Gabriella,
Kim HyeYeon,
Balamotis Michael A.,
Seligman Len M.,
Nakamoto Robert K.,
Bowie James U.
Publication year - 2009
Publication title -
protein science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.353
H-Index - 175
eISSN - 1469-896X
pISSN - 0961-8368
DOI - 10.1002/pro.39
Subject(s) - recombinant dna , protein expression , selection (genetic algorithm) , escherichia coli , biology , membrane protein , expression (computer science) , genetics , computational biology , microbiology and biotechnology , chemistry , membrane , gene , computer science , artificial intelligence , programming language
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.