Cell-Free Protein Synthesis Technology in NMR High-Throughput Structure Determination

Abstract

This chapter describes the current implementation of the cell-free translation platform developed at the Center for Eukaryotic Structural Genomics (CESG) and practical aspects of the production of stable isotope-labeled eukaryotic proteins for NMR structure determination. Protocols are reported for the use of wheat germ cell-free translation in small-scale screening for the level of total protein expression, the solubility of the expressed protein, and the success in purification as predictive indicators of the likelihood that a protein may be obtained in sufficient quantity and quality to initiate structural studies. In most circumstances, the small-scale reactions also produce sufficient protein to permit bioanalytical and functional characterizations. The protocols incorporate the use of robots specialized for small-scale cell-free translation, large-scale protein production, and automated purification of soluble, His(6)-tagged proteins. The integration of isotopically labeled proteins into the sequence of experiments required for NMR structure determination is outlined, and additional protocols for production of integral membrane proteins in the presence of either detergents or unilamellar liposomes are presented.

Apoglobin Stability Is the Major Factor Governing both Cell-free and in Vivo Expression of Holomyoglobin

“…A small scale holoMb purification protocol was modified from the protocol for His 6 -tagged proteins (S. Makino, University of Wisconsin, Center for Eukaryotic Structural Genomics) (40). Zn 2ϩ -chelating resin (GBiosciences) was added into the wells of a MultiScreen HTS HV 0.45-m filter plate (Millipore) to purify 10 Ϫ10 to 10 Ϫ11 mol of soluble holoMb generated from each cell-free translation reaction (total volume Ϸ230 l).…”

“…A 96-well U bottom plate (Grenier Bio One) was affixed below the 96-well MultiScreen HTS HV filter plate containing the Zn 2ϩ resin for collection of wash buffers and then the eluted holoMb, following centrifugation of the plates at 3640 rpm for 1 min (40). The pH and salt concentration of the purification wash and elution buffers were optimized as described below to reduce nonspecific binding of eukaryotic proteins on the Zn 2ϩ resin (41).…”

Spinning proteins, the faster, the better?

“…Here, expression systems which allow the production of deuterated proteins directly in H2O, as can be done using cell-free systems, [62][63][64] are highly desirable, as they incorporate deuterated amino acids, while still synthesizing the protein in a protonated environment. On the other hand, as small amounts of protein are best measured using as small as possible rotors (to optimize the possible signal-to noise ratio), fast MAS would actually not only profit from the powerful selective labeling possibilities inherent in cell-free expression, but would indeed open the door for the study of proteins interesting from a medical point of view.…”

Wheat-germ cell-free production of prion proteins for solid-state NMR structural studies

“…The implementation of these methods also obviates the need for cell harvesting, cell lysis, and shorten protein purification. Finally, and most importantly, many different labeling schemes can be achieved with cell-free methods [15,24,25,29,33]. As cell extracts for in vitro expression are costly, we have developed in our laboratory the production of wheat-germ cell-free extracts, based on protocols developed by Endo and coworkers over the past years [34,35].…”

“…Cell-free synthesis is an alternative technique which offers several advantages over cell-based methods, and has started to be used in structural biology including NMR [24][25][26]. Firstly, it allows expression of proteins that would interfere with cell physiology.…”