title: Engineering membrane protein overproduction in Escherichia coli.
abstract: Membrane proteins play a fundamental role in human disease and therapy, but suffer from a lack of structural and functional information compared to their soluble counterparts. The paucity of membrane protein structures is primarily due to the unparalleled difficulties in obtaining detergent-solubilized membrane proteins at sufficient levels and quality. We have developed an in vitro evolution strategy for optimizing the levels of detergent-solubilized membrane protein that can be overexpressed and purified from recombinant Escherichia coli. Libraries of random mutants for nine membrane proteins were screened for expression using a novel implementation of the colony filtration blot. In only one cycle of directed evolution were significant improvements of membrane protein yield obtained for five out of nine proteins. In one case, the yield of detergent-solubilized membrane protein was increased 40-fold.
authors.affiliation: Department of Biochemistry and Biophysics, Stockholm University, S-106 91 Stockholm, Sweden.
authors.forename: Daniel
authors.initials: D
authors.lastname: Martinez Molina
authors.forename: Tobias
authors.initials: T
authors.lastname: Cornvik
authors.forename: Said
authors.initials: S
authors.lastname: Eshaghi
authors.forename: Jesper Z
authors.initials: JZ
authors.lastname: Haeggström
authors.forename: Pär
authors.initials: P
authors.lastname: Nordlund
authors.forename: Marina Ignatushchenko
authors.initials: MI
authors.lastname: Sabet
mesh_terms: D003001:Cloning, Molecular; D003902:Detergents; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D015723:Gene Library; D006801:Humans; D008565:Membrane Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D016296:Mutagenesis; D015202:Protein Engineering / Q000379:methods*; D017433:Protein Structure, Secondary
chemical_list: D003902:Detergents; D008565:Membrane Proteins