Preparation of Multiple Site-Specific Mutant Proteins for NMR Studies by PCR-Directed Cell-Free Protein Synthesis.
 

Preparation of Multiple Site-Specific Mutant Proteins for NMR Studies by PCR-Directed Cell-Free Protein Synthesis.

Preparation of Multiple Site-Specific Mutant Proteins for NMR Studies by PCR-Directed Cell-Free Protein Synthesis.

Methods Mol Biol. 2014;1118:169-87

Authors: Ozawa K, Qi R

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, ?CTS:?c16.


PMID: 24395416 [PubMed - in process]



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