title: A systematic approach to increase the efficiency of membrane protein production in cell-free expression systems.
abstract: High amounts of membrane protein samples are needed for structural or functional analysis and a first bottleneck is often to obtain sufficient production efficiencies. The reduced complexity of protein production in cell-free expression systems results in a frequent correlation of efficiency problems with the essential transcription/translation process. We present a systematic tag variation strategy for the rapid improvement of cell-free expression efficiencies of membrane proteins based on the optimization of translation initiation. A small number of rationally designed short expression tags is attached via overlap PCR to the 5-prime end of the target protein coding sequence. The generated pool of DNA templates is analyzed in a cell-free expression screen and the most efficient template is selected for further preparative scale protein production. The expression tags can be minimized to only a few codons and no further impact on the coding sequence is required. The complete process takes only few days and the synthesized PCR fragments can be used directly as templates for preparative scale cell-free reactions. The strategy is exemplified with the production of a set of G-protein coupled receptors and yield improvements of up to 32-fold were obtained. All proteins were finally synthesized in amounts sufficient for further quality optimization and initial crystallization screens.
authors.affiliation: Institute of Biophysical Chemistry, Centre for Biomolecular Magnetic Resonance, J.W. Goethe-University, Frankfurt-am-Main, Germany.
authors.forename: Stefan
authors.initials: S
authors.lastname: Haberstock
authors.forename: Christian
authors.initials: C
authors.lastname: Roos
authors.forename: Yvette
authors.initials: Y
authors.lastname: Hoevels
authors.forename: Volker
authors.initials: V
authors.lastname: Dötsch
authors.forename: Gisela
authors.initials: G
authors.lastname: Schnapp
authors.forename: Alexander
authors.initials: A
authors.lastname: Pautsch
authors.forename: Frank
authors.initials: F
authors.lastname: Bernhard
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001483:Base Sequence; D002846:Chromatography, Affinity; D003062:Codon; D004926:Escherichia coli; D015870:Gene Expression; D010957:Plasmids / Q000235:genetics; D043562:Receptors, G-Protein-Coupled / Q000096:biosynthesis* / Q000302:isolation & purification; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000302:isolation & purification; D013347:Subcellular Fractions
chemical_list: D003062:Codon; D043562:Receptors, G-Protein-Coupled; D011993:Recombinant Fusion Proteins