Takashi Yabuki

Cell-free production and stable-isotope labeling of milligram quantities of proteins.

We have improved the productivity of an Escherichia coli cell‐free protein synthesis system. First, creatine phosphate and creatine kinase were used as the energy source regeneration system, and the other components of the reaction mixture were optimized. Second, the E. coli S30 cell extract was condensed by dialysis against a polyethylene glycol solution to increase the rate of synthesis. Third, during the protein synthesis, the reaction mixture was dialyzed against a low‐molecular‐weight substrate solution to prolong the reaction. Thus, the yield of chloramphenicol acetyltransferase was raised to 6 mg/ml of reaction mixture. Stable‐isotope labeling of a protein with 13C/15N‐labeled amino acids for NMR spectroscopy was achieved by this method.

Structural genomics projects in Japan.

Two major structural genomics projects exist in Japan. The oldest, the RIKEN Structural Genomics Initiative, has two major goals: to determine bacterial, mammalian, and plant protein structures by X-ray crystallography and NMR spectroscopy and to perform functional analyses with the target proteins. The newest, the structural genomics project at the Biological Information Research Center, focuses on human membrane proteins.

Preparation of Escherichia coli cell extract for highly productive cell-free protein expression

AbstractAs structural genomics and proteomics research has become popular, the importance of cell-free protein synthesis systems has been realized for high-throughput expression. Our group has established a high-throughput pipeline for protein sample preparation for structural genomics and proteomics by using cell-free protein synthesis. Among the many procedures for cell-free protein synthesis, the preparation of the cell extract is a crucial step to establish a highly efficient and reproducible workflow. In this article, we describe a detailed protocol for E. coli cell extract preparation for cell-free protein synthesis, which we have developed and routinely use. The cell extract prepared according to this protocol is used for many of our cell-free synthesis applications, including high-throughput protein expression using PCR-amplified templates and large-scale protein production for structure determinations. abbreviations 2-mEt — 2-mercaptoethanol; AcCoA — acetyl coenzyme A; BL21 CP — BL21 codon-plus RIL; CAT — chloramphenicol acetyl transferase; CK — creatine kinase; Cm — chloramphenicol; DEPC — diethylpyrocarbonate; CP — creatine phosphate; DTNB — 5,5′-dithiobis-2-nitrobenzoic acid; DTT — dithiothreitol; Folinic acid — L(−)-5-formyl-5,6,7,8-tetrahydrofolic acid; KGlu — polyethyleneglycol; PEG — potassium glutamate; PEP — phospho-enolpyruvate; PK — pyruvate kinase.

An unnatural base pair for incorporating amino acid analogs into proteins

An unnatural base pair of 2-amino-6-(2-thienyl)purine (denoted by s) and pyridin-2-one (denoted by y) was developed to expand the genetic code. The ribonucleoside triphosphate of y was site-specifically incorporated into RNA, opposite s in a template, by T7 RNA polymerase. This transcription was coupled with translation in an Escherichia coli cell-free system. The yAG codon in the transcribed ras mRNA was recognized by the CUs anticodon of a yeast tyrosine transfer RNA (tRNA) variant, which had been enzymatically aminoacylated with an unnatural amino acid, 3-chlorotyrosine. Site-specific incorporation of 3-chlorotyrosine into the Ras protein was demonstrated by liquid chromatography–mass spectrometry (LC-MS) analysis of the products. This coupled transcription–translation system will permit the efficient synthesis of proteins with a tyrosine analog at the desired position.

An engineered Escherichia coli tyrosyl–tRNA synthetase for site-specific incorporation of an unnatural amino acid into proteins in eukaryotic translation and its application in a wheat germ cell-free system

Tyrosyl–tRNA synthetase (TyrRS) from Escherichia coli was engineered to preferentially recognize 3-iodo-l-tyrosine rather than l-tyrosine for the site-specific incorporation of 3-iodo-l-tyrosine into proteins in eukaryotic translation systems. The wild-type TyrRS does not recognize 3-iodo-l-tyrosine, because of the bulky iodine substitution. On the basis of the reported crystal structure of Bacillus stearothermophilus TyrRS, three residues, Y37, Q179, and Q195, in the l-tyrosine-binding site were chosen for mutagenesis. Thirty-four single amino acid replacements and 16 of their combinations were screened by in vitro biochemical assays. A combination of the Y37V and Q195C mutations changed the amino acid specificity in such a way that the variant TyrRS activates 3-iodo-l-tyrosine 10-fold more efficiently than l-tyrosine. This engineered enzyme, TyrRS(V37C195), was tested for use in the wheat germ cell-free translation system, which has recently been significantly improved, and is now as productive as conventional recombinant systems. During the translation in the wheat germ system, an E. coli suppressor tRNATyr was not aminoacylated by the wheat germ enzymes, but was aminoacylated by the E. coli TyrRS(V37C195) variant with 3-iodo-l-tyrosine. After the use of the 3-iodotyrosyl–tRNA in translation, the resultant uncharged tRNA could be aminoacylated again in the system. A mass spectrometric analysis of the produced protein revealed that more than 95% of the amino acids incorporated for an amber codon were iodotyrosine, whose concentration was only twice that of l-tyrosine in the translation. Therefore, the variant enzyme, 3-iodo-l-tyrosine, and the suppressor tRNA can serve as an additional set orthogonal to the 20 endogenous sets in eukaryotic in vitro translation systems.

Solution Structure of an Arabidopsis WRKY DNA Binding Domain

The WRKY proteins comprise a major family of transcription factors that are essential in pathogen and salicylic acid responses of higher plants as well as a variety of plant-specific reactions. They share a DNA binding domain, designated as the WRKY domain, which contains an invariant WRKYGQK sequence and a CX4–5CX22–23HXH zinc binding motif. Herein, we report the NMR solution structure of the C-terminal WRKY domain of the Arabidopsis thaliana WRKY4 protein. The structure consists of a four-stranded β-sheet, with a zinc binding pocket formed by the conserved Cys/His residues located at one end of the β-sheet, revealing a novel zinc and DNA binding structure. The WRKYGQK residues correspond to the most N-terminal β-strand, kinked in the middle of the sequence by the Gly residue, which enables extensive hydrophobic interactions involving the Trp residue and contributes to the structural stability of the β-sheet. Based on a profile of NMR chemical shift perturbations, we propose that the same strand enters the DNA groove and forms contacts with the DNA bases.

Dual amino acid-selective and site-directed stable-isotope labeling of the human c-Ha-Ras protein by cell-free synthesis

We developed two methods for stable-isotope labeling of proteins by cell-free synthesis. Firstly, we applied cell-free synthesis to the dual amino acid-selective 13C-15N labeling method, originally developed for in vivo systems by Kainosho and co-workers. For this purpose, we took one of the advantages of a cell-free protein synthesis system; the amino acid-selective stable-isotope labeling is free of the isotope scrambling problem. The targets of selective observation were Thr35 and Ser39 in the ‘effector region’ (residues 32–40) of the Ras protein complexed with the Ras-binding domain of c-Raf-1 (Raf RBD) (the total molecular mass is about 30 kDa). Using a 15-mL Escherichia coli cell-free system, which was optimized to produce about 0.4 mg of Ras protein per 1-mL reaction, with 2 mg each of DL-[13C′]proline and L-[15N]threonine, we obtained about 6 mg of Ras protein. As the Pro–Thr sequence is unique in the Ras protein, the Thr35 cross peak of the Ras•Raf RBD complex was unambiguously identified by the 2D 1H–15N HNCO experiment. The Ser39 cross peak was similarly identified with the [13C′]Asp/[15N]Ser-selectively labeled Ras protein. There were no isotope scrambling problems in this study. Secondly, we have established a method for producing a milligram quantity of site-specifically stable-isotope labeled protein by a cell-free system involving amber suppression. The E. coli amber suppressor tRNATyr_CUA (25 mg) was prepared by in vitro transcription with T7 RNA polymerase. We aminoacylated the tRNATyr_CUA transcript with purified E. coli tyrosyl-tRNA synthetase, using 2 mg of l-[15N]tyrosine. In the gene encoding the Ras protein, the codon for Tyr32 was changed to an amber codon (TAG). This template DNA and the [15N]Tyr-tRNATyr_CUA were reacted for 30 min in 30 mL of E. coli cell-free system. The subsequent purification yielded 2.2 mg of [15N]Tyr32-Ras protein. In the 1H–15N HSQC spectrum of the labeled Ras protein, only one cross peak was observed, which was unambiguously assigned to Tyr32.

Solution Structure of the B3 DNA Binding Domain of the Arabidopsis Cold-Responsive Transcription Factor RAV1

The B3 DNA binding domain is shared amongst various plant-specific transcription factors, including factors involved in auxin-regulated and abscisic acid–regulated transcription. Herein, we report the NMR solution structure of the B3 domain of the Arabidopsis thaliana cold-responsive transcription factor RAV1. The structure consists of a seven-stranded open β-barrel and two α-helices located at the ends of the barrel and is significantly similar to the structure of the noncatalytic DNA binding domain of the restriction enzyme EcoRII. An NMR titration experiment revealed a DNA recognition interface that enabled us to propose a structural model of the protein–DNA complex. The locations of the DNA-contacting residues are also likely to be similar to those of the EcoRII DNA binding domain.

Regioselective Carbon–Carbon Bond Formation in Proteins with Palladium Catalysis; New Protein Chemistry by Organometallic Chemistry

Palladium‐catalyzed reactions have contributed to the advancement of many areas of organic chemistry, in particular, the synthesis of organic compounds such as natural products and polymeric materials. In this study, we have used a Mizoroki–Heck reaction for site‐specific carbon–carbon bond formation in the Ras protein. This was performed by the following two steps: 1) the His6‐fused Ras protein containing 4‐iodo‐L‐phenylalanine at position 32 (iF32‐Ras‐His) was prepared by genetic engineering and 2) the aryl iodide group on the iF32‐Ras‐His was coupled with vinylated biotin in the presence of a palladium catalyst. The biotinylation was confirmed by Western blotting and liquid chromatography–mass spectrometry (LC‐MS). The regioselectivity of the Mizoroki–Heck reaction was furthermore confirmed by LC‐MS/MS analysis. However, in addition to the biotinylated product (bF32‐Ras‐His), a dehalogenated product (F32‐Ras‐His) was detected by LC‐MS/MS. This dehalogenation resulted from the undesired termination of the Mizoroki–Heck reaction due to steric and electrostatic hindrance around residue 32. The biotinylated Ras showed binding activity for the Ras‐binding domain as its downstream target, Raf‐1, with no sign of decomposition. This study is the first report of an application of organometallic chemistry in protein chemistry.

Solution Structure of the SEA Domain from the Murine Homologue of Ovarian Cancer Antigen CA125 (MUC16)

Human CA125, encoded by the MUC16 gene, is an ovarian cancer antigen widely used for a serum assay. Its extracellular region consists of tandem repeats of SEA domains. In this study we determined the three-dimensional structure of the SEA domain from the murine MUC16 homologue using multidimensional NMR spectroscopy. The domain forms a unique α/β sandwich fold composed of two α helices and four antiparallel β strands and has a characteristic turn named the TY-turn between α1 and α2. The internal mobility of the main chain is low throughout the domain. The residues that form the hydrophobic core and the TY-turn are fully conserved in all SEA domain sequences, indicating that the fold is common in the family. Interestingly, no other residues are conserved throughout the family. Thus, the sequence alignment of the SEA domain family was refined on the basis of the three-dimensional structure, which allowed us to classify the SEA domains into several subfamilies. The residues on the surface differ between these subfamilies, suggesting that each subfamily has a different function. In the MUC16 SEA domains, the conserved surface residues, Asn-10, Thr-12, Arg-63, Asp-75, Asp-112, Ser-115, and Phe-117, are clustered on the β sheet surface, which may be functionally important. The putative epitope (residues 58-77) for anti-MUC16 antibodies is located around the β2 and β3 strands. On the other hand the tissue tumor marker MUC1 has a SEA domain belonging to another subfamily, and its GSVVV motif for proteolytic cleavage is located in the short loop connecting β2 and β3.