An economical method for producing stable-isotope labeled proteins by the E. coli cell-free system
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- Yokoyama, J., Matsuda, T., Koshiba, S. et al. J Biomol NMR (2010) 48: 193. doi:10.1007/s10858-010-9455-3
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Improvement of the cell-free protein synthesis system (CF) over the past decade have made it one of the most powerful protein production methods. The CF approach is especially useful for stable-isotope (SI) labeling of proteins for NMR analysis. However, it is less popular than expected, partly because the SI-labeled amino acids used for SI labeling by the CF are too expensive. In the present study, we developed a simple and inexpensive method for producing an SI-labeled protein using Escherichia coli cell extract-based CF. This method takes advantage of endogenous metabolic conversions to generate SI-labeled asparagine, glutamine, cysteine, and tryptophan, which are much more expensive than the other 16 kinds of SI-labeled amino acids, from inexpensive sources, such as SI-labeled algal amino acid mixture, SI-labeled indole, and sodium sulfide, during the CF reaction. As compared with the conventional method employing 20 kinds of SI-labeled amino acids, highly enriched uniform SI-labeling with similar labeling efficiency was achieved at a greatly reduced cost with the newly developed method. Therefore, our method solves the cost problem of the SI labeling of proteins using the CF.
KeywordsCell-free protein synthesisIn vitro translationStable-isotope labelingMetabolic conversion
Recent 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). With respect to stable-isotope (SI) labeling of proteins for NMR analysis, both highly enriched and amino acid-selective labeling have been enabled by many kinds of improvements (Kigawa et al. 1995; Matsuda et al. 2007; Morita et al. 2004; Ozawa et al. 2004; Wu et al. 2006). A comparison of the cell-based and CF-based methods for protein production indicated that the CF had advantages in terms of NMR-based high-throughput sample screening and labor cost (Tyler et al. 2005). Optimization efforts have focused on making CF simpler and more economical (Calhoun and Swartz 2005; Kim et al. 2006; Kim et al. 2007; Liu et al. 2008). Many structures of functional protein domains were determined in large-scale structural genomics projects by using the high-throughput robotic CF and high performance NMR spectroscopy (Aoki et al. 2009; Kigawa et al. 2004; Vinarov et al. 2004; Yabuki et al. 2007; Yokoyama 2003). The CF is now considered as a suitable method for membrane protein production for structure analysis (Shimono et al. 2009; Sobhanifar et al. 2010). The stereo-array isotope labeling (SAIL) method (Kainosho et al. 2006), which is expected to expand the molecular size limit of NMR spectroscopy, was achieved by using the CF. However, the CF is still not very popular in NMR applications, partly due to the high cost of SI-labeled amino acids.
Generally, uniformly SI-labeled amino acids are produced from an acid hydrolysate of a stable-isotopically enriched algal protein biomass. However, L-asparagine (Asn), L-glutamine (Gln), L-cysteine (Cys), and L-tryptophan (Trp) are sensitive to acid hydrolysis and thus are not present in the hydrolysate (Hansen et al. 1992). These four acid-sensitive amino acids are prepared individually by using more complicated and time-consuming methods, and consequently are much more expensive than the other 16 amino acids contained in the hydrolysate. Instead of using costly purified SI-labeled amino acids, a relatively inexpensive SI-labeled algal amino acid mixture (AAM) can be used to reduce the labeling cost, but the expensive SI-labeled Asn, Gln, Cys, and Trp are still required (Kigawa et al. 1999; Kigawa et al. 2004). Therefore, the cost of SI labeling using the CF strongly depends on that of the SI-labeled, acid-sensitive amino acids.
Materials and methods
Cell-free protein synthesis and NMR sample preparation
For chloramphenicol acetyltransferase (CAT) synthesis, the pk7-CAT plasmid (Kim et al. 1996) was used. The CAT productivity was calculated as previously described (Kigawa et al. 2004). For green fluorescence protein (GFP) synthesis, the pGFPS1 plasmid, harboring a gene encoding the mutant protein GFPS1 (Seki et al. 2008), was used. The composition of the E. coli cell extract-based CF reaction using D-glutamate was previously described (Matsuda et al. 2007). The batch mode of the CF was performed as described (Kigawa et al. 1999). The dialysis mode of the CF, using the small-scale dialysis unit, and the affinity purification of the product protein were accomplished as described (Matsuda et al. 2007). All of the proteins for NMR measurements were prepared using the dialysis mode method, with 3 mL of internal solution and 30 mL of external solution (Kigawa et al. 2007). Uniformly 15N-labeled Ras(Y32 W) protein (Matsuda et al. 2007; Yamasaki et al. 1994) (BMRB: 10051) (Ras(Y32 W)/20AAM) was synthesized with 3 mg/mL of uniformly 15N-labeled algal AAM (Isotec, Ohio, USA), which contained 16 kinds of amino acids, except for Asn, Gln, Cys, and Trp, 1 mM each of 15N-Asn (Isotec), 15N-Gln (Isotec), 15N-Cys (Taiyo Nippon Sanso, Tokyo, Japan), and 0.3 mM 15N-Trp (Taiyo Nippon Sanso) as the standard condition. The 15N-labeled Ras(Y32 W)/16AAM was synthesized with 3 mg/mL of 15N-labeled algal AAM, 27 mM 15N-labeled ammonium acetate (Isotec), 0.3 mM 15N-labeled indole (Cambridge Isotope Laboratories, Inc., CIL, Massachusetts, USA), 1 mM sodium sulfide (Na2S) nonahydrate (Wako Pure Chemicals, Osaka, Japan), which was converted into H2S in the CF reaction, and 0.2 mM pyridoxal 5′-phosphate (PLP) (Wako Pure Chemicals), which was added for the enhancement of PLP-requiring enzymes, and 0.1 mM acetyl coenzyme A (AcCoA). Indole, a precursor of Trp, was dissolved in 50% ethanol to prepare a 30 mM stock solution, and Na2S, a precursor of Cys, was freshly dissolved in water before use. Stock solutions of the inhibitors, amino-oxyacetate (AOA: Wako Pure Chemicals) (John et al. 1978; Morita et al. 2004), L-methionine sulfoximine (MS: Nakalai Tesuque, Kyoto, Japan) (Manning et al. 1969), S-methyl-L-cysteine sulfoximine (SMCS), synthesized as described previously (Koizumi et al. 1999), and D-malate (Wako Pure Chemicals) (Falzone et al. 1988), were prepared by dissolving them in water at a concentration of 200 mM each. Uniformly 15N-labeled ubiquitin-associated (UBA) domains (Leu655-Ser715 of ubiquitin carboxyl-terminal hydrolase 5 (SWISS-PROT: P45974, PDB: 2DAG, BMRB: 11173), UBA/20AAM and UBA/16AAM, were synthesized using the same conditions as for Ras(32 W)/20AAM and Ras(32 W)/16AAM, respectively. The side-chain selectively 15N-labeled UBA domain was synthesized with 1.5 mM each of 18 non-labeled amino acids (the usual 20 except for Gln and Asn) and 27 mM 15N-labeled ammonium acetate.
Amino acid composition analysis in the cell-free protein synthesis reaction
A 20 μL aliquot of the batch mode of the CF reaction without template DNA was mixed with 20 μL of 5% trichloroacetic acid, and then centrifuged for 10 min at 12,000×g to remove the precipitated proteins, as described (Calhoun and Swartz 2006). A 10 μL aliquot of the resultant supernatant was mixed with 70 μL of borate buffer and 20 μL of the derivatizing reagent, 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate, from the AccQ-Tag kit (Waters, Massachusetts, USA), and the solution was incubated for 10 min at 55°C in an aluminum block heater. The analyte was applied to a UPLC amino acids analysis system with an AccQ-Tag Ultra Column (2.1 × 100 mm), and was analyzed using the AccQ-Tag method (Waters), in order to investigate amino acid fluctuation during the CF reaction.
Labeled Ras(Y32 W) proteins were concentrated to 0.5 mM in 20 mM sodium phosphate buffer (pH 6.5), containing 100 mM NaCl, 5 mM MgCl2, 5 mM d-DTT, 0.01% NaN3, and 10% D2O. Labeled UBA domains were concentrated to 0.5 mM in 20 mM d-Tris–HCl (pH 7.0), containing 100 mM NaCl, 1 mM d-DTT, 0.02% NaN3, and 10% D2O. Two-dimensional 1H-15N HSQC spectra (Kay et al. 1992) were measured at 25°C on AVANCE 600 MHz or 700 MHz spectrometers equipped with a CryoProbe (Bruker BioSpin, Karlsruhe, Germany). Data were processed using TopSpin 2.1 (Bruker BioSpin).
Results and discussion
CF reaction with a reduced set of amino acids
The metabolic conversion of amino acids in the CF reaction was further investigated using the UPLC amino acids analysis system with AccQ-Tag, which could analyze the composition of the 20 kinds of amino acids within 10 min. Asn and Gln were detected after a 1 h incubation at 37°C, in the batch mode of the CF reaction solution lacking template DNA and including 1.5 mM 20AAM-Asn and 20AAM-Gln, respectively (Fig. 2b). Similarly, Trp was detected in the reaction solution with 1.5 mM 20AAM-Trp, 0.3 mM indole, and 0.2 mM PLP. On the other hand, only a small amount of Cys was detected in the reaction solution with 1.5 mM 20AAM-Cys, 1 mM Na2S, and 0.1 mM AcCoA (Fig. 2b), probably because the amount of Cys in the CF reaction solution was maintained at a low level by the feedback regulation of SAT by Cys (Kredich and Tomkins 1966; Mino et al. 1999). These results indicate that Asn, Gln, Trp, and Cys can be synthesized from their precursors by endogenous metabolism in the CF reaction solution.
Finally, using the optimized conditions with 3 mg/mL of 15N-labeled AAM, 0.3 mM 15N-indole, 1 mM Na2S, 0.2 mM PLP, 0.1 mM AcCoA, and 27 mM 15N-labeled ammonium acetate, the productivity for both CAT and GFP became almost equal to that under standard conditions (Fig. 2c, d).
Side-chain amide selective labeling of Asn and Gln residues
Asn and Gln residues play important structural roles in protein–protein and protein-substrate interactions, because their side-chain amide groups can act as both hydrogen bond acceptors and donors on the surface of proteins or in the active site of enzymes. For example, observation of the side-chain amide groups of Asn and Gln residues was useful to understand the interaction of hen egg-white lysozyme with its substrate (Higman et al. 2004).
Uniformly 15N-labeled proteins synthesized with the optimized conditions and NMR measurements
Over the past few years, various improvements and optimizations have been made for the SI-labeling of proteins using the CF. The E. coli S30 extract (Spirin et al. 1988) for the CF contains not only essential substances for translation but also various housekeeping enzymes, such as nucleases (Seki et al. 2009; Yang et al. 1980), proteases, and amino acid transaminases (Ozawa et al. 2004). The major enzymes involved in amino acid degradation in the CF extract were identified, and their deficient mutant strains were created for the CF (Calhoun and Swartz 2006). Some inhibitors to suppress the metabolic pathways related to the isotope dilution were also investigated (Morita et al. 2004; Ozawa et al. 2004). These studies indicated that a thorough understanding of amino acid metabolism in the CF was especially important for establishing the CF with low amino acid consumption. Therefore, a technique to monitor during the CF reaction, using a UPLC amino acids analysis system with AccQ-Tag, was established in this study, which greatly contributed to clarifying the metabolic amino acid degradation and conversion in the CF. In almost all of the previous studies on cost reduction of SI labeling, amino acid metabolism was inhibited and/or removed, in order to suppress the consumption of expensive amino acids. On the other hand, in the present study, amino acid metabolism was utilized to generate expensive amino acids from inexpensive sources during the CF reaction. Our approach successfully achieved both convenient and cost-effective SI-labeling of proteins using the CF. The combination of suppressing undesirable metabolic enzymes by the chemical inhibitor and/or the genetic engineering and activation of useful enzymes for metabolic conversion/generation presented in this study will enable more efficient consumption of SI-labeled compounds, and therefore, better cost-effective SI-labeling of proteins with the CF. This will certainly offer more opportunities to produce SI-labeled proteins for NMR analysis using the CF.
We would like to thank Naoya Tochio, Eiko Seki, and Natsuko Matsuda for valuable suggestions and discussions. We would also like to thank Kenji Fukuda and Mariko Seki for help with the amino acid analysis. This work was supported in part by the Collaborative Development of Innovative Seeds programs (Potentiality verification stage and Practicability verification stage) of the Japan Science and Technology Agency.