Application Note 19

In vitro Protein Synthesis of Perdeuterated Proteins for NMR Studies

Touraj Etezady-Esfarjani,¹ Sebastian Hiller,¹ Cristina Villalba,¹ Kurt Wüthrich^1,2

1. Institute for Molecular Biology and Biophysics, ETH Zurich, 8093 Zurich, Switzerland
2. Department of Molecular Biology and Skaggs Institute for Chemical Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037 USA

It is well documented that high levels of deuteration are indispensable for solution NMR studies of polypeptides in structures of sizes above 40 kDa (Fiaux, et al., 2002; LeMaster, 1989; Pachter, et al., 1992). In addition to studies on protein structure and dynamics, obtaining a perdeuterated background is of potential interest for studies of protein functions using residue-selective stable isotope labeling. Unfortunately, the yield of expressed protein in vivo in ²H₂O-based media is often significantly lower than that obtained in H₂O-based growth media, even after lengthy “training” of the cells. 

Cell-free protein synthesis can provide for efficient incorporation of selectively labeled amino acids into polypeptide chains in situations where in vivo protein expression typically results in isotope scrambling or isotope dilution (Kigawa, et al., 1995; Ozawa, et al., 2004; Waugh, 1996), and its use can extend to cytotoxic proteins, such as proteases or apoptosis-related proteins (Adrain, et al., 2006). So far, cell-free protein synthesis protocols for uniformly deuterated proteins typically yield low nonuniform deuteration levels. This application note summarizes work (Etazady, et al., 2007) using cell-free synthesis methods employing both H₂O-based and ²H₂O-based E. coli cell extracts for designed, nonuniform incorporation of ²H, ¹⁵N-labeled amino acids. NMR results are presented for the 14 kDa FK 506-binding protein (FKBP) and GroEL, an 800 kDa E. coli chaperonine oligomeric protein with 14 identical subunits (Xu, et al., 1997).

Read more by downloading the application note.