Big Title
Our research
~Analysis to synthesis~
We are studying diverse biological mechanisms and designing novel bio-processes.
Efficient cell-free protein synthesis and its applications
The cell-free protein synthesis (or in vitro protein synthesis) is a biosynthesis of proteins from DNA or mRNA templates using cell extract containing a complete translation machinery such as ribosome, translation factors, tRNA etc. Compared to the protein synthesis in the living cells, the cell-free protein synthesis (CFPS) 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 a template.
We have been aiming to further improve the efficiency of CFPS system and have developed new application technologies that utilise the features of this system. One of these is the Ecobody technology, described below. Another application technology relying on CFPS is in vitro single-molecule display used for evolution of peptides and enzymes, described in the next section.
Monoclonal antibodies (mAbs) are used in a variety of applications, including food testing, clinical testing, pharmaceuticals and biosensors. However, development of mAbs are costly and time-consuming and its resources are limited to a certain number of animal species, and have the disadvantage that there are many molecules that are difficult to produce. Our research group has established a technology that enables the rapid synthesis and evaluation of Fab antibodies using a cell-free protein synthesis system by selecting B cells from human and immunized animal blood and then amplifying the gene for a monoclonal antibody molecule from a single B cell. The Fab antibodies obtained can be produced in required quantities by using microorganisms such as E. coli in an integrated monoclonal antibody selection and production system (referred to as the Ecobody method) (see figure). Based on this technology, Dr. Nakano and Dr. Ojima-Kato founded the startup company iBody Ltd. in 2018, which operates independently of our laboratory.
Currently, our laboratory is working on the advancement and simplification of Ecobody technology by making full use of B cell selection methods, ribosome display, bioinformatics and microbial protein production systems, with aim at constructing a system for easy search, evaluation and production of useful monoclonal antibodies
Responsible staff: Dr. Hideo Nakano and Dr. Teruyo Ojima-Kato
In vitro single-molecule display
for directed evolution of enzymes and enzyme substrates
Directed evolution is a method in protein engineering that mimics the process of natural selection to obtain proteins with desired properties, such as specificity, stability, high turnover, etc. Enzymes in nature have evolved to catalyze chemical reactions at a certain rate, specificity, etc., which are not always suitable for applications we need them for. The properties of enzymes can be improved by protein engineering, where directed evolution plays a key role. However, generated diversity requires efficient screening of large libraries with less investment of time, labor, and cost. cDNA display is an in vitro screening method developed to select functional proteins from large combinatorial libraries (~10^14) in a matter of days, using simple and robust protocols. It utilizes cell-free protein synthesis and covalent linkage between genotype and phenotype. Our project aims to develop a novel platform for the directed evolution of enzymes based on cDNA display technology coupled with next-generation sequencing and bioinformatics. Current targets include oxidoreductases, lipid-modifying enzymes, and bond-forming enzymes.
Responsible staff: Dr. Jasmina Damnjanovic
Translation enhancement by nascent peptide chain
and its application to protein production
Protein production in microorganisms such as E. coli and yeast, as well as in animal ans plant cells, is becoming increasingly important for biotechnology research and industry, and for a sustainable society. Proteins are produced in all living organisms through mRNA translation and folding, but some proteins are difficult to express. Our group has found that the production of some proteins that are difficult to express in E. coli can be increased by adding a short peptide tag to the N-terminus of the protein. The nascent peptide tag itself may also have a function to improve translation efficiency. In this study, we are screening for new peptide tags that can promote translation and analyzing the mechanism by ribosome profiling using E. coli, yeast, animal cells, and cell- free protein synthesis systems. Ultimately, we aim to understand the mechanism of translation promotion by nascent peptides and to apply the new knowledge to protein production by microorganisms and animal cells. Our research outcomes are expected to be utilized in various fields, including the discovery and functional analysis of various proteins (antibodies, enzymes, differentiation factors, etc.), production of biopharmaceutical and food-related proteins.
Responsible staff: Dr. Teruyo Ojima-Kato
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.
