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

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:τ_c16.