|Efficient cell-free protein synthesis and its application
The cell-free protein synthesis (or in vitro protein synthesis) is a biosynthesis system of proteins from DNA or mRNA templates using cell extract containing various translation machineries such as ribosome, translation factors, tRNA etc. Comparing to the living cell-based protein synthesis, the cell-free protein synthesis enables; 1) Correct protein folding by controlling the reaction conditions (temperature, redox, addition of co-factors or chaperons), even if the target protein is not able to fold correctly in living cells, 2) Synthesis of toxic proteins, which can not be synthesized in the living cells, 3) Quick synthesis of the target proteins within a few hours, and 4) Use of PCR amplified DNA directly as the templates, thereby the synthesis of various proteins in a high throughput manner. We have attempt even greater efficiency of the cell-free protein synthesis and have established novel screening systems by using it. For example, we have developed a simple method for protein library construction by combining the cell-free system and single-molecule PCR technique (SIMPLEX) and have applied it for creation novel tailor-made enzymes. Moreover, we have also developed an epoch-making method called single-cell RT-PCR-linked in vitro expression (SICREX), which enables the high-throughput generation and screening of monoclonal antibodies.
|Enzymatic synthesis of value-added lipids
Almost all the chemical reactions in living cells are catalyzed by enzymes. Enzymes are able to work under mild conditions on specific positions of specific substrates. We are studying the syntheses of useful products by using these excellent enzymesﾕ properties. The current projects deal with the enzymatic syntheses of value-added lipids by lipid-related enzymes such as lipases, phospholipases and etc. Moreover, we are also doing researches on development of new analytical methods of lipids (e.g. HPLC-assisted separation of lipid isomers). These lipid products are of interest for the industrial applications in the fields of food, cosmetics and medicals.
|Development of biomolecular-interaction screening method with emulsion PCR
Recently, many kinds of novel methods have been developed for functional biomolecular selection from “molecular library”. These selection techniques are classified as either “in vivo” or “in vitro”. The in vitro approach which needs not living cell enables rapid and easy-to-use selection. Our laboratory has developed an epoch-making method, solid-phase single-molecule PCR in w/o emulsions. Single-molecule DNA amplifications are carried out on microbeads in w/o emulsions. As a result, hundreds of DNA molecules derived from single-template are immobilized on the each microbead. Thus, by this PCR, DNA library is converted to “beads library” which enables rapid and easy-to-use in vitro selection. By the addition of fluorescent-label protein to the beads library and FACS screening, the “bait protein” interactive-DNA or the “prey protein” coding DNA will be enriched preferentially. The in vitro selection system would be applied to various research fields.
|Genome-wide identification of genes regulated by transcription factors (TFs) by a combinatorial analysis using high-throughput sequencing and bioinformatics
Transcription factors (TFs) are proteins which regulate gene expression. In particular, DNA binding type TFs play a central role in Transcription regulation. We have developed a novel analysis pipeline for genome-wide identification of genes regulated by TFs by a combinatorial analysis using genomic systematic evolution of ligands by exponential enrichment (gSELEX)-Seq, RNA-Seq and bioinformatics. gSELEX-Seq provides the information of in vitro binding sites of a target TF on a genome-wide scale. Conversely, RNA-Seq read out the information of the differentially expressed genes (DEGs) modulated by the target TF in vivo. This analytical pipeline is a robust and versatile system for genome-wide identification of the genes that are regulated by a target TF. Now we are trying the identification of the target genes regulated by various TFs using this system.