Preparation of multiple site-specific mutant proteins for NMR studies by PCR-directed cell-free protein synthesis

Authors

Kiyoshi Ozawa, University of WollongongFollow
Ruhu Qi, Australian National University

RIS ID

86413

Publication Details

Ozawa, K. & Qi, R. (2014). Preparation of multiple site-specific mutant proteins for NMR studies by PCR-directed cell-free protein synthesis. Methods in Molecular Biology, 1118 169-187.

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 1H,15N-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 1H,15 N-NMR spectra obtained from 12 Ile/Ala substituted mutants of the 40 kDa protein complex, αCTS:τc16.