Takanori Kigawa, Ph.D
RIKEN Systems and Structural /Biology Center
1-7-22 Suehiro-cho Tsurumi,
Yokohama, 230-0045

Introduction

In recent years, technological advances have made it possible to produce a wide variety of eukaryotic and prokaryotic proteins in large quantities by using the cell-free protein synthesis system (CF). In addition to the conventional E. coli, rabbit and wheat germ cell extract-based systems, the new CF is based on eukaryotic cell extracts that have been developed. There has been a growing need for protein expression methods optimized for a high-throughput format to prepare an extremely large number of protein samples in an efficient and rapid way. The CF is especially suitable for such needs because it is easily adapted to automated and/or high-throughput procedures.

Structural genomics and proteomics projects have adopted the CF (for example, (Yokoyama, 2003)). The CF has widely become regarded as one of the most useful protein expression methods. In our group, the Escherichia coli cell extract-based coupled transcription-translation CF is now one of the most routinely used expression methods. More than 1,300 NMR structures and 250 X-ray structures of proteins/protein domains have already been determined using the CF as the protein production method.

Large-scale Production

The low productivity problem of the CF has been solved by the modification of the reaction conditions (for example, (Yabuki et al., 1998)) and the development of the continuous method (Spirin et al., 1988), which is a major technological breakthrough in the CF. The continuous exchange CF, that is, the dialysis-mode CF is the most popular format for both small-scale and large-scale productions (Kigawa et al., 2007). The CF reaction mixture in the dialysis tube is dialyzed against the external solution containing substrates of transcription and translation, such as amino acids and nucleotide triphosphates.

Protein production usually continues for several hours to overnight, and the productivity reaches more than 7 mg protein/mL of reaction mixture by ten hours of incubation, for CAT protein (Kigawa et al., 2007). Reaction scale is from 1 mL internal /10 mL external format to 9 mL internal /90 mL external format with almost the same productivity per reaction mixture volume if the proportion of the surface area of the dialysis tube to the reaction volume is kept at optimum. One of the major advantages of the CF is that PCR-amplified linear DNA fragments can be achieved without any cloning procedures. A simple modification of the cell extract preparation protocol, just by altering the cultivation temperature of the cells to a moderately lower range (20–34 °C), drastically reduced the linear DNA degradation activity in the cell extract. As a result, even the large-scale production using the dialysis system can be performed with PCR-amplified template with sufficient productivity (Seki et al., 2008).

Automation

As mentioned above, the CF is easily adapted to automated and/or high-throughput procedures. The fully automated CF-based protein production system has been developed in various formats. One of the systems can perform the PCR reaction for template generation (Yabuki et al., 2007), the batch mode CF reaction (30 µL), and, optionally, the fluorescence measurement of GFP-fused product for 768 samples at the same time within 9 hours (Kigawa et al., 2007).

Another system that integrates the dialysis mode CF (1 mL of internal solution) and the affinity purification can prepare 96 kinds of purified proteins in milligram quantities within a half day (Aoki et al., 2009). Nearly 20,000 protein samples using the dialysis CF with the PCR-amplified linear DNA template were prepared, and then their NMR spectra were successfully measured (Kigawa et al., 2007).

Uniform Stable-Isotope Labeling

Amino acid selective or uniform labeling can be, in principle, achieved by simply replacing the amino acid(s) of interest in the CF reaction mixture with labeled one(s). The CF requires potassium ions for efficient translation and potassium L-glutamate is usually used as the potassium ion source (Kigawa et al., 1995).

However, high concentration of potassium L-glutamate is not suitable for stable-isotope labeling (uniform or glutamate-selective). Alternatively, the CF that uses potassium D-glutamate as the potassium ion source (D-Glu system) was developed to achieve highly productive CF suitable for stable-isotope labeling (Matsuda et al., 2007). More than one thousand uniformly ¹³C/¹⁵N-labeled proteins from higher eukaryotes, such as human, mouse and Arabidopsis, have been prepared by using the D-Glu system. The NMR spectra of these proteins were of sufficient quality for structural analyses. The stereo-array isotope labeling (SAIL) method (Kainosho et al., 2006), which is expected to expand the molecular size limit of NMR spectroscopy, is one of the applications of ‟uniform” stable-isotope labeling using the CF.

Purified SI-labeled amino acids are conventionally used for uniform labeling (Kigawa, 2010). Instead of using them, a less expensive SI-labeled algal amino acid mixture (AAAM), produced from an acid hydrolysate of an SI-enriched algal protein biomass, can be used to reduce the labeling cost. However, the SI-labeled　asparagine, glutamine, cysteine and tryptophan, which are not contained in the hydrolysate and much more expensive than the other 16 amino acids, are still required (Kigawa et al., 1999). Therefore, the cost of SI-labeling using the CF strongly depends on that of the SI-labeled acid-sensitive 4 amino acids. These 4 amino acids can be generated from inexpensive sources, such as SI-labeled AAAM, SI-labeled indole and sodium sulfide, during the CF reaction by taking advantage of endogenous metabolic conversions (Yokoyama et al., 2010).

As compared with the conventional method, employing 20 kinds of purified SI-labeled amino acids, highly enriched uniform SI-labeling with similar labeling efficiency was achieved at a greatly reduced cost. Perdeuteration is generally required for analyses of larger proteins. Extremely high-level perdeuteration, which is generally difficult using the conventional In Vivo production, was achieved by using the CF with perdeuterated amino acids (Etezady-Esfarjani et al., 2007). However, all of the reagents, including the E. coli cell extract, were prepared with ²H²O, because the deuterium dilution of the non-labile hydrogens at the α-and β-positions in certain amino acids occurred in the conventional ¹H²O-based CF. It was reported that non-labile hydrogen exchange in amino acids was catalyzed by pyridoxal 5’-phosphate (PLP) requiring enzymes.

By simply adding inhibitors of the PLP-requiring enzymes, the ¹H²O-based CF with perdeuterated amino acids can prepare the perdeuterated protein (Yokoyama et al., 2011).

Amino-Acid Selective Stable-Isotope Labeling

Amino-acid selective SI-labeling has been widely used to solve the problems of low sensitivity and spectral overlapping in NMR spectroscopy, and moreover, it has become crucial for tackling higher molecular weight proteins and their complexes.

The CF especially enables the selective labeling of almost any type of amino acid with modest isotopic scrambling and dilution (for example (Kigawa et al., 1995), except for certain amino acids, such as aspartic acid, asparagine, serine and alanine. By simply suppressing endogenous metabolic reactions in the CF with their chemical inhibitors; aminooxyacetate, D-malate, L-methionine sulfoximine, S-methyl-L-cysteine sulfoximine, 6-diazo-5-oxo-L-norleucine, and 5-diazo-4-oxo-L-norvaline, precise and complete amino-acid selective labeling was achieved (Yokoyama et al., 2011). This SI-labeling technique, with no scrambling and dilution of labels, will significantly contribute to NMR analyses of large and complicated biological systems because the precision and completeness of the SI-labeling are beneficial to newly developed SI-labeling strategies. Suppressing amino acid metabolism also contributes to the increased labeling efficiency and thus reduces the cost of SI-labeling to some extent.

The methyl-selective protonation of isoleucine, leucine and valine residues has also expanded the molecular size limit of NMR spectroscopy (Tugarinov and Kay, 2003). Recently, structure determination of a nearly 400-residue protein was successfully achieved using the methyl-selective protonation samples prepared by the CF.

Site-directed Stable-Isotope Labeling

Site-directed stable-isotope labeling drastically simplifies the observation and resonance-assignment procedures for a specified amino acid residue of particular interest, and will therefore be useful, for example, in analyzing local structures of large proteins and protein-protein interactions. The site-directed incorporation of unnatural amino acids has been achieved by the CF involving amber (codon UAG) suppression (Noren et al., 1989). By using almost the same procedures, site-directed (Tyr³²) incorporation of SI-labeled tyrosine into Ras protein was achieved and then its ¹H–¹⁵N HSQC spectrum was successfully measured (Yabuki et al., 1998).

The site-directed incorporation of an SI-labeled amino acid other than tyrosine is possible if the aminoacylated suppressor tRNA is available.

Membrane Protein

The CF is now considered as a suitable method for membrane protein production for structure analysis in order to overcome major restrictions of conventional In Vivo production (Shimono et al., 2009, Sobhanifar et al., 2010, Nguyen et al., 2010). In addition to the general advantages of the CF, such as avoidance of toxicity and proteolysis, the membrane proteins can be directly integrated into membrane cotranslationally for functional folding.

Future Prospects

The CF still has enough room for modification and improvement in order to achieve more sophisticated SI-labeling of proteins. In combination with the novel NMR methods, it is expected to further expand the potential of biomolecular NMR.

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