title: High-yield Escherichia coli-based cell-free expression of human proteins.
abstract: Production of sufficient amounts of human proteins is a frequent bottleneck in structural biology. Here we describe an Escherichia coli-based cell-free system which yields mg-quantities of human proteins in N-terminal fusion constructs with the GB1 domain, which show significantly increased translation efficiency. A newly generated E. coli BL21 (DE3) RIPL-Star strain was used, which contains a variant RNase E with reduced activity and an excess of rare-codon tRNAs, and is devoid of lon and ompT protease activity. In the implementation of the expression system we used freshly in-house prepared cell extract. Batch-mode cell-free expression with this setup was up to twofold more economical than continuous-exchange expression, with yields of 0.2-0.9 mg of purified protein per mL of reaction mixture. Native folding of the proteins thus obtained is documented with 2D [(15)N,(1)H]-HSQC NMR.
authors.affiliation: Institute of Molecular Biology and Biophysics, ETH Zurich, 8093 Zurich, Switzerland.
authors.forename: Erich
authors.initials: E
authors.lastname: Michel
authors.forename: Kurt
authors.initials: K
authors.lastname: Wüthrich
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D002457:Cell Extracts; D002474:Cell-Free System; D003001:Cloning, Molecular / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D005822:Genetic Vectors; D006801:Humans; D007553:Isotope Labeling; D019906:Nuclear Magnetic Resonance, Biomolecular; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D002457:Cell Extracts; C034284:IgG Fc-binding protein, Streptococcus; D011993:Recombinant Fusion Proteins