HOHSAKA Laboratory
<Major Research Areas>Genetic engineering, Biomolecular engineering, Chemical biology

Biosynthesis of proteins containing
nonnatural amino acids by
using extended genetic code

Research activity

1Incorporation of nonnatural amino acids into proteins by using four-base codons

　　Proteins are made up of only 20 types of amino acids. If nonnatural amino acids can be used for protein synthesis, possibilities of protein designs are greatly expanded. We have developed a novel technology allowing us to introduce chemically synthesized nonnatural amino acids into desired positions of proteins. This technology is accomplished by using four-base codons as extended genetic code encoding nonnatural amino acids.
　　Applying this technology, nonnatural proteins with artificial functions such as photoreactivity have been investigated. In addition, novel nonnatural amino acids and transfer RNAs have been developed to improve this technology. Through collaboration with bio-ventures, industrial applications of the technology are now in progress.

2Development of novel methodologies for protein structural and functional analyses

　　The protein synthesizing system allowing the incorporation of nonnatural amino acids is applied to structural and functional analyses of proteins. Particularly, position-specific incorporation of fluorescent amino acids has been developed to label proteins and investigate the protein functions. The fluorescence labeling method is expected to become a useful tool for analysis of disease-related proteins and drug screening against the proteins.

3Development of novel technologies utilizing potential biological functions

　　There will be a lot of potential biological functions available as unique biotechnologies. Four-base decoding and thermostable DNA polymerase used in PCR and DNA sequencing are examples of the potential biofunctions. We are developing novel technologies utilizing potential biofunctions for contributing to the progress of life science.

Equipment

DNA sequencer, Fluorescence image analyzer, real-time PCR, Fluorescence spectrophotometer, Ultra-low volume UV-Vis spectrophotometer, ESI-Mass, HPLC, Surface plasmon resonance biosenser

The main research achievements in the past five years

1. M. Miura, N. Muranaka, R. Abe, T. Hohsaka, Incorporation of fluorescent-labeled non-α-amino carboxylic acids into the N-terminus of proteins in response to amber initiation codon, Bull. Chem. Soc. Jpn., 83, 546-553 (2010).
2. I. Iijima, and T. Hohsaka, Position-specific incorporation of fluorescent non-natural amino acids into maltose-binding protein for detection of ligand binding by FRET and fluorescence quenching, ChemBioChem, 10, 999-1006 (2009).
3. T. Watanabe, Y. Miyata, R. Abe, N. Muranaka, and T. Hohsaka, N-terminal specific fluorescence labeling of proteins through incorporation of fluorescent hydroxy acid and subsequent ester cleavage, ChemBioChem, 9, 1235-1242 (2008).
4. H. Taira, Y. Matsushita, K. Kojima, K. Shiraga, T. Hohsaka, Comprehensive screening of amber suppressor tRNAs suitable for incorporation of non-natural amino acids in a cell-free translation system, Biochem. Biophys. Res. Commun., 374, 304-308 (2008).
5. D. Kajihara, R. Abe, I. Iijima, C. Komiyama, M. Sisido, and T. Hohsaka, FRET analysis of protein conformational change through position-specific incorporation of fluorescent amino acids, Nature Methods, 3, 923-929 (2006).