RESEARCH

Protein synthesis (translation) is carried out by the ribosome with numerous translation factors. We have successfully established the in vitro protein expression system using purified human ribosomes and translation factors. This system is now used to explore how the human translation is regulated and dysregulated.

We are creating cell-like vessels (artificial eukaryotic cells) by assembling cellular components. We enclose the human reconstituted translation system in the cell-like vessels, and express therein various proteins such as the Huntingtin-gene product, which forms aggregates in the nerve cells and causes Huntington's disease. We are thus creating in vitro model systems for diseases using these cell-like vessels.

Fig 1 Translation of CoV2
The genome RNA of the coronavirus comprises of approximately 3x104 nucleotides, and upon translation two large polyproteins are produced. Whereas the smaller polyprotein is generated by the authentic translation, the larger one, which contains the RNA polymerase, is synthesized through an event called frameshift. The RNA polymerase amplifies the genome RNA and synthesizes sub-genomic RNAs, leading to amplification of the virus.

Fig 2 Reporter assay system for CoV2 frameshift
An expression vector for the in vitro translation is constructed by incorporating a nucleotide fragment involved in the frameshift event of CoV2. This vector is devised such that EGFP (a fluorescent protein) can be produced upon the frameshift event.

Fig 3 Inhibitor screening for CoV2 frameshift
The in vitro translation system programmed with the CoV2-EGFP expression vector is distributed to numerous plate wells, where different chemicals are input in advance. If a fluorescent score from a well is reduced, the well is judged to contain a chemical that potentially inhibits the CoV2 frameshift.