title: Preparation of multiple site-specific mutant proteins for NMR studies by PCR-directed cell-free protein synthesis.
abstract: Cell-free protein synthesis (CFPS) offers a fast and inexpensive approach to selectively label proteins with isotopes that can then be detected by nuclear magnetic resonance (NMR) spectroscopy directly in the translation mixture. We describe a PCR-based approach for production of protein-coding circularized DNA templates that can be expressed in Escherichia coli extract in CFPS dialysis system. This approach typically yields target protein concentrations close to 1 mg/mL, which is sufficient for subsequent analysis by 2D (1)H,(15)N-NMR. Furthermore, this PCR-based technique also enables parallel preparation of mutant proteins in a high-throughput mode, enabling rapid assignments of NMR signals. This chapter describes the general CFPS protocol that we used to rapidly assign residue-specific cross peaks from 2D (1)H,(15 N-NMR) spectra obtained from 12 Ile/Ala substituted mutants of the 40 kDa protein complex, αCTS:τc16.
authors.affiliation: School of Chemistry, University of Wollongong, Wollongong, NSW, Australia.
authors.forename: Kiyoshi
authors.initials: K
authors.lastname: Ozawa
authors.forename: Ruhu
authors.initials: R
authors.lastname: Qi
mesh_terms: D002457:Cell Extracts; D002474:Cell-Free System; D004926:Escherichia coli / Q000201:enzymology; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000096:biosynthesis* / Q000235:genetics*; D019906:Nuclear Magnetic Resonance, Biomolecular; D016133:Polymerase Chain Reaction; D014176:Protein Biosynthesis; D013698:Templates, Genetic
chemical_list: D002457:Cell Extracts; D050505:Mutant Proteins