Takaaki Kojima

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.

Microbeads Display of Proteins Using Emulsion PCR and Cell-Free Protein Synthesis

We developed a method for coupling protein to its coding DNA on magnetic microbeads using emulsion PCR and cell‐free protein synthesis in emulsion. A PCR mixture containing streptavidin‐coated microbeads was compartmentalized by water‐in‐oil (w/o) emulsion with estimated 0.5 template molecules per droplet. The template molecules were amplified and immobilized on beads via bead‐linked reverse primers and biotinylated forward primers. After amplification, the templates were sequentially labeled with streptavidin and biotinylated anti‐glutathione S‐transferase (GST) antibody. The pool of beads was then subjected to cell‐free protein synthesis compartmentalized in another w/o emulsion, in which templates were coupled to their coding proteins. We mixed two types of DNA templates of Histidine6 tag (His6)‐fused and FLAG tag‐fused GST in a ratio of 1:1,000 (His6: FLAG) for use as a model DNA library. After incubation with fluorescein isothiocyanate (FITC)‐labeled anti‐His6 (C‐term) antibody, the beads with the His6 gene were enriched 917‐fold in a single‐round screening by using flow cytometry. A library with a theoretical diversity of 106 was constructed by randomizing the middle four residues of the His6 tag. After a two‐round screening, the randomized sequences were substantially converged to peptide‐encoding sequences recognized by the anti‐His6 antibody.

Directed evolution of angiotensin II-inhibiting peptides using a microbead display

Angiotensin II (ang II), an octapeptide (DRVYVHPF), can regulate blood pressure by binding specifically to its receptor, AT1. A peptide (VVIVIY) in the first transmembrane of AT1 has been found, via peptide array technology, to have an affinity for ang II. In this study, the peptide P2, which contained the VVIVIY sequence, was mutated and screened using microbead display technology that utilized emulsion PCR and cell-free protein synthesis. After one round of screening, the binding activities of collected mutants were estimated using flow cytometry and a peptide array. Two of these exhibited improved association rate constants to ang II, compared to the P2 peptide.

Novel screening system for protein–protein interactions by bimolecular fluorescence complementation in Saccharomyces cerevisiae

For high-throughput screening of protein-protein interactions, we have developed a novel yeast screening system using Bimolecular fluorescence complementation (BiFC). Two yeast plasmids, in which genes of heterodimerized peptides LZA and LZB were each fused with those of non-fluorescent half fragments of Kusabira-Green mutant (mKG2), were transformed into a- and α-type yeast, respectively. Mating of them gave a library, which was screened by following green fluorescence resulted from LZA-LZB interaction. The method showed potential ability to detect the positive clones from a model library, in which green-fluorescent and non-fluorescent yeast was mixed in a ratio of 1:675.

Expression Profiles of Polyhydroxyalkanoate Synthesis-Related Genes in Paracoccus denitrificans

A facultative methylotrophic bacterium, Paracoccus denitrificans can synthesize polyhydroxyalkanoate acids (PHA) from various alcohols. Recently, six genes, phaA, B, C, P, R, and Z, related to PHA synthesis have been cloned and characterized. PHA synthesis and the expression of phaA, B, C, P, R, and Z in P. denitrificans were examined at the transcriptional and translational levels under both nitrogen-sufficient and nitrogen-deficient conditions. The results showed that PHA synthesis is not regulated at the mRNA or protein level in phaA, B, and C. We also observed the condensation of acetyl-coenzyme A (acetyl-CoA) in the cells by high-performance liquid chromatography (HPLC). The results suggest that the amount of acetyl-CoA would regulate PHA synthesis. Finally, we discuss a possible regulation mechanism for PHA synthesis in P. denitrificans.

Ultra-High-Throughput Screening of an In Vitro-Synthesized Horseradish Peroxidase Displayed on Microbeads Using Cell Sorter

The C1a isoenzyme of horseradish peroxidase (HRP) is an industrially important heme-containing enzyme that utilizes hydrogen peroxide to oxidize a wide variety of inorganic and organic compounds for practical applications, including synthesis of fine chemicals, medical diagnostics, and bioremediation. To develop a ultra-high-throughput screening system for HRP, we successfully produced active HRP in an Escherichia coli cell-free protein synthesis system, by adding disulfide bond isomerase DsbC and optimizing the concentrations of hemin and calcium ions and the temperature. The biosynthesized HRP was fused with a single-chain Cro (scCro) DNA-binding tag at its N-terminal and C-terminal sites. The addition of the scCro-tag at both ends increased the solubility of the protein. Next, HRP and its fusion proteins were successfully synthesized in a water droplet emulsion by using hexadecane as the oil phase and SunSoft No. 818SK as the surfactant. HRP fusion proteins were displayed on microbeads attached with double-stranded DNA (containing the scCro binding sequence) via scCro-DNA interactions. The activities of the immobilized HRP fusion proteins were detected with a tyramide-based fluorogenic assay using flow cytometry. Moreover, a model microbead library containing wild type hrp (WT) and inactive mutant (MUT) genes was screened using fluorescence-activated cell-sorting, thus efficiently enriching the WT gene from the 1:100 (WT:MUT) library. The technique described here could serve as a novel platform for the ultra-high-throughput discovery of more useful HRP mutants and other heme-containing peroxidases.

Role of disulfide bond isomerase DsbC, calcium ions, and hemin in cell-free protein synthesis of active manganese peroxidase isolated from Phanerochaete chrysosporium.

A cell-free protein synthesis system can produce various types of proteins directly from DNA templates such as PCR products, and therefore attracts great attention as an alternative protein synthesis system especially for high-throughput functional screening of proteins. Here, we report successful expression of active Phanerochaete chrysosporium manganese peroxidase (MnP) in an Escherichia coli cell-free protein synthesis system, wherein reaction conditions such as the concentrations of hemin, calcium ions, and disulfide bond isomerase were optimized to increase the solubility and activity of the synthesized enzyme. Moreover, cell-free synthesized MnP purified using the hemagglutinin tag showed higher specific activity than the commercial wild-type enzyme, suggesting that the cell-free system can be used as a preparative method for efficient synthesis of disulfide bond-containing metalloenzymes such as MnP. We believe that our system is a solid foundation for the development of a high-throughput screening method for the directed evolution of these enzymes.

In vitro generation of rabbit anti-Listeria monocytogenes monoclonal antibody using single cell based RT-PCR linked cell-free expression systems.

Rabbit monoclonal antibodies (mAbs) have advantages over mouse antibodies in diagnostics and biotechnological applications owing to higher affinity and specificity.We developed a platform to generate rabbit mAbs by a novel monoclonal antibody generation method named “Single-Cell Reverse Transcription-PCR linked in vitro-Expression (SICREX)” system. In this method,we use single-cell based RT-PCR followed by sequential PCR steps of mAb genes and subsequent cell-free protein synthesis (CFPS) by using linear DNA fragments of mAbs. This platform enables the rapid generation and evaluation of mAbs derived from antigen-specific single B cells in the peripheral blood of immunized animals without mammalian cell cultivation. In this study, the antigen used was a food-borne gram-positive pathogen, Listeria monocytogenes, that is known to cause serious infection. Three active mAbs in CFPS were obtained by constructing the single chain of variable fragment (scFv) form. These scFvs were produced in the cytoplasm of E. coli Shuffle T7 Express strain as an active form and used for further investigation.

Comprehensive Analysis of the DNA-Binding Specificity of an Aspergillus nidulans Transcription Factor, AmyR, Using a Bead Display System

The in vitro DNA binding profile of Aspergillus nidulans transcription factor AmyR was analyzed by a novel approach employing a genetic library of beads and flow cytometry analysis. An artificial library with 22 randomized nucleotides was constructed and subjected to a protein-DNA binding reaction with MalE-tagged AmyR. DNA fragments with potential AmyR-binding sites were labeled with fluorescence-conjugated antibody to be enriched by flow cytometry through 5 rounds of successive selection. Finally, a binding motif with a single CGG triplet was obtained from DNA fragments showing weak AmyR binding, while another motif with dual CGG triplets was discovered with stronger binding fragments. An informative motif, CGGNNNTTTNTCGG, was found to exist only in the promoter region of highly AmyR-dependent genes. These results suggest that this system is a powerful tool for the rapid and comprehensive analysis of the binding preferences of transcription factors.

Immobilization of proteins onto microbeads using a DNA binding tag for enzymatic assays

A novel DNA-binding protein tag, scCro-tag, which is a single-chain derivative of the bacteriophage lambda Cro repressor, has been developed to immobilize proteins of interest (POI) on a solid support through binding OR consensus DNA (ORC) that is tightly bound by the scCro protein. The scCro-tag successfully bound a transglutaminase 2 (TGase 2) substrate and manganese peroxidase (MnP) to microbeads via scaffolding DNA. The resulting protein-coated microbeads can be utilized for functional analysis of the enzymatic activity using flow cytometry. The quantity of bead-bound proteins can be enhanced by increasing the number of ORCs. In addition, proteins with the scCro-tag that were synthesized using a cell-free protein synthesis system were also immobilized onto the beads, thus indicating that this bead-based system would be applicable to high-throughput analysis of various enzymatic activities.