Yasuaki Kawarasaki

PCR amplification from single DNA molecules on magnetic beads in emulsion: application for high-throughput screening of transcription factor targets

We have developed a novel method of genetic library construction on magnetic microbeads based on solid-phase single-molecule PCR in a fine and robust water-phase compartment formed in water-in-oil (w/o) emulsions. In this method, critically diluted DNA fragments were distributed over the emulsion as templates, where beads crosslinked with multiple primers and other PCR components were encapsulated to form multiple reaction compartments. The delivered DNA was then amplified and covalently immobilized on the beads in parallel, within individual compartments, to construct a genetic library on beads (GLOBE), which was readily applicable to a genomewide global scanning of genetic elements recognized by a defined DNA-binding protein. We constructed a GLOBE of Paracoccus denitrificans and selected gene beads that were bound to the His-tagged transcription factor PhaR by flow cytometry. As a result of flow cytometry screening with an anti-His fluorescent antibody, the PhaR target fragments were enriched 1200-fold from this library with this system. Therefore, this system is a powerful tool for analyzing the transcription network on a genomewide scale.

Export Pathway Selectivity of Escherichia coli Twin Arginine Translocation Signal Peptides

The Escherichia coli genome encodes at least 29 putative signal peptides containing a twin arginine motif characteristic of proteins exported via the twin arginine translocation (Tat) pathway. Fusions of the putative Tat signal peptides plus six to eight amino acids of the mature proteins to three reporter proteins (short-lived green fluorescent protein, maltose-binding protein (MBP), and alkaline phosphatase) and also data from the cell localization of epitope-tagged full-length proteins were employed to determine the ability of the 29 signal peptides to direct export through the Tat pathway, through the general secretory pathway (Sec), or through both. 27/29 putative signal peptides could export one or more reporter proteins through Tat. Of these, 11 signal peptides displayed Tat specificity in that they could not direct the export of Sec-only reporter proteins. The rest (16/27) were promiscuous and were capable of directing export of the appropriate reporter either via Tat (green fluorescent protein, MBP) or via Sec (PhoA, MBP). Mutations that conferred a ≥+1 charge to the N terminus of the mature protein abolished or drastically reduced routing through the Sec pathway without affecting the ability to export via the Tat pathway. These experiments demonstrate that the charge of the mature protein N terminus affects export promiscuity, independent of the effect of the folding state of the mature protein.

Crystal structure of glucansucrase from the dental caries pathogen Streptococcus mutans.

Glucansucrase (GSase) from Streptococcus mutans is an essential agent in dental caries pathogenesis. Here, we report the crystal structure of S. mutans glycosyltransferase (GTF-SI), which synthesizes soluble and insoluble glucans and is a glycoside hydrolase (GH) family 70 GSase in the free enzyme form and in complex with acarbose and maltose. Resolution of the GTF-SI structure confirmed that the domain order of GTF-SI is circularly permuted as compared to that of GH family 13 α-amylases. As a result, domains A, B and IV of GTF-SI are each composed of two separate polypeptide chains. Structural comparison of GTF-SI and amylosucrase, which is closely related to GH family 13 amylases, indicated that the two enzymes share a similar transglycosylation mechanism via a glycosyl-enzyme intermediate in subsite -1. On the other hand, novel structural features were revealed in subsites +1 and +2 of GTF-SI. Trp517 provided the platform for glycosyl acceptor binding, while Tyr430, Asn481 and Ser589, which are conserved in family 70 enzymes but not in family 13 enzymes, comprised subsite +1. Based on the structure of GTF-SI and amino acid comparison of GTF-SI, GTF-I and GTF-S, Asp593 in GTF-SI appeared to be the most critical point for acceptor sugar orientation, influencing the transglycosylation specificity of GSases, that is, whether they produced insoluble glucan with α(1-3) glycosidic linkages or soluble glucan with α(1-6) linkages. The structural information derived from the current study should be extremely useful in the design of novel inhibitors that prevent the biofilm formation by GTF-SI.

High-throughput, cloning-independent protein library construction by combining single-molecule DNA amplification with in vitro expression.

A novel, cloning-independent strategy for construction of protein libraries has been developed and demonstrated experimentally. A pool of genes is prepared and thereafter extensively diluted to give one molecule of DNA per well. Each individual molecule is amplified separately by polymerase chain reaction (single-molecule PCR) yielding a PCR library. Subsequently, the PCR library is directly transformed into a protein library by means of in vitro coupled transcription/translation. Amounts of DNA produced by the single-molecule PCR were equal and uniformity of amounts of successively in vitro synthesized proteins, which were critical for quantitative comparison among clones in the library, was better than that of the classical in vivo expression system. Here, we describe a library of anti-human serum albumin single-chain antibodies (anti-HSA-scFv) originating from a monoclonal anti-HSA-scFv which was constructed and screened in order to demonstrate its real practicability. Application of the strategy described for high-throughput generation and screening of protein libraries is discussed.

Highly productive cell-free protein synthesis system using condensed wheat-germ extract

In order to increase the productivity of a cell-free protein synthesis system using wheat-germ extract, the extract was highly condensed by polyethylene glycol precipitation, which greatly increased both the final protein concentration and the initial synthesis rate of translation. However, the translational reaction using the condensed extract ceased earlier than the reaction using the uncondensed extract did, because ribonuclease and phosphatase activities, which destroyed mRNA and lessened the ATP level, respectively, were also condensed. Adding Cu2+ to the condensed translational system was found to reduce these activities resulting in a prolonged reaction with a high initial rate. Finally, 120 μg of dihydrofolate reductase protein from Escherichia coli was obtained in 1 ml of reaction mixture in 3 h. In addition, the initial protein synthesis rate was 70 μg ml−1 h−1, which was about 10 times higher than that of a conventional uncondensed extract.

Analyzing a dipeptide library to identify human dipeptidyl peptidase IV inhibitor

Human dipeptidyl peptidase IV (hDPPIV) inhibitors provide an effective strategy for the treatment of type 2 diabetes. Because certain peptides are known to act as hDPPIV inhibitors, a dataset of possible peptides with their inhibition intensities will facilitate the development of functional food for type 2 diabetes. In this study, we examined a total of 337 dipeptides with respect to their hDPPIV inhibitory effects. Amino acid residues at N-termini dominated their inhibition intensities. Particularly highly inhibitory dipeptides discovered included the following novel dipeptides: Thr-His, Asn-His, Val-Leu, Met-Leu, and Met-Met. Using our dataset, prime candidates contributing to the hDPPIV inhibitory effect of soy protein hydrolyzates were successfully identified. Possible dietary proteins potentially able to produce particularly highly hDPPIV inhibitory peptides are also discussed on the basis of the dataset.

Analysing the substrate multispecificity of a proton-coupled oligopeptide transporter using a dipeptide library

Peptide uptake systems that involve members of the proton-coupled oligopeptide transporter (POT) family are conserved across all organisms. POT proteins have characteristic substrate multispecificity, with which one transporter can recognize as many as 8,400 types of di/tripeptides and certain peptide-like drugs. Here we characterize the substrate multispecificity of Ptr2p, a major peptide transporter of Saccharomyces cerevisiae, using a dipeptide library. The affinities (Ki) of di/tripeptides toward Ptr2p show a wide distribution range from 48 mM to 0.020 mM. This substrate multispecificity indicates that POT family members have an important role in the preferential uptake of vital amino acids. In addition, we successfully establish high performance ligand affinity prediction models (97% accuracy) using our comprehensive dipeptide screening data in conjunction with simple property indices for describing ligand molecules. Our results provide an important clue to the development of highly absorbable peptides and their derivatives including peptide-like drugs.

Phosphatase-immunodepleted cell-free protein synthesis system

A cell-free protein synthesis system using wheat-germ extract was improved by a novel approach involving selective removal of endogenous phosphatase, which reduces both the duration and the rate of translation by hydrolyzing ATP and GTP, from the translational reaction. Immunodepletion of the phosphatases by the antibodies raised against the major one of the wheat-germ phosphatase isozymes removed 20-40% of ATP-hydrolysis activity from the wheat-germ extract, and thereby prolonged the reaction period of translation. Moreover, the condensation of the phosphate-immunodepleted extract by polyethylene glycol (PEG) precipitation and the addition of copper ions, which was known to inhibit phosphatase and nuclease activity, increased the protein synthesis more than two-fold compared with the reaction using control IgG-treated condensed extract.

Trp-Arg-Xaa tripeptides act as uncompetitive-type inhibitors of human dipeptidyl peptidase IV.

Human dipeptidyl peptidase IV (hDPPIV, alternative name: CD26) inhibitors provide an effective strategy for the treatment of type 2 diabetes. Recently, our research group discovered a non substrate-mimic inhibitory dipeptide, Trp-Arg, by the systematic analysis of a dipeptide library. In the present study, a tripeptide library Trp-Arg-Xaa (where Xaa represents any amino acid) was analyzed to investigate the interactions of peptidergic inhibitors with hDPPIV. Trp-Arg-Glu showed the highest inhibitory effect toward hDPPIV (Ki=130 μM). All of the tested 19 Trp-Arg-Xaa tripeptides showed unique uncompetitive-type inhibition. The inhibition mechanism of Trp-Arg-Xaa is discussed based on the crystal structure of hDPPIV. The information obtained by this study suggests a novel concept for developing hDPPIV inhibitory peptides and drugs.

Chimeric Gene Library Construction by a Simple and Highly Versatile Method Using Recombination‐Dependent Exponential Amplification

A simple and efficient method for the construction of chimeric gene libraries termed RDA‐PCR (recombination‐dependent exponential amplification polymerase chain reaction) was developed by modifying polymerase chain reaction. A chimeric gene library is generated from homologous parental genes with additional primer‐annealing sequences at their “heads” and “tails”. Two primers (“skew primers”) are designed to exclusively anneal to either the heads of maternal genes or the tails of paternal genes. During the RDA‐PCR, short annealing/extension periods facilitate homologous recombination. The chimeric sequences can be exponentially amplified to form the chimeric gene library, whereas parental sequences without crossovers are not amplified. As a model, we constructed a chimeric gene library of yellow and green fluorescent protein (yfp and gfp, respectively). The crossover point profile of RDA‐PCR clones was compared with those obtained by (modified) family shuffling. PCR restriction fragment polymorphism (PCR‐RFLP) analysis of the RDA‐PCR clones showed a high content of chimeric genes in the library, whereas family shuffling required the modification using skew primers for selective enrichment of chimeric sequences. PCR‐RFLP analysis also indicated that the crossover points of RDA‐PCR chimeras were distributed over the entire protein‐coding region. Moreover, as few as 2 bp of the continual identity of nucleotides were found at the crossover points at high frequency (30% of the tested clones), suggesting that RDA‐PCR resulted in a higher diversity in crossover points than family shuffling.