Inventors

Kensaku Sakamoto, Takahito Mukai (Expanded Genetic Code System Research Team)

Background

Non-natural proteins refer to proteins that have amino acids other than 20 canonical amino acids (non-natural amino acids) or α-hydroxy acids in place of amino acid residues at defined sites. Synthesis of non-natural protein promises to diversify proteins beyond the current limitations in protein engineering with natural amino acids, and will be a basic technology to produce novel proteins endowed with new physiological activities, catalyst activities, structures or functions. Unfortunately, efficient incorporation of a non-natural amino acid is currently limited to one site of a protein, which hinders free design and creation of non-natural proteins. In general, an amber codon (UAG codon), one of the stop codons, is used to introduce non-natural amino acid for production of non-natural proteins. Several methods have been published so far for efficient incorporation of non-natural amino acids. For example in the case of E. coli, a method to weaken activity of translation termination factor, and a method to substitute all amber codons on E. coli genome with other stop codons, and destroy genes encoding translation termination factor, have been proposed. But in the former method, if we want to incorporate more than one non-natural amino acid, the efficiency of incorporation is markedly poor, and the latter method lacks versatility because it requires extensive modification of gene: even E. coli has 314 amber codons that need substitution with stop codons.

Summary

We developed a method to produce E. coli that can produce proteins having more non-natural amino acids in a highly efficient manner, without compromising the growth rate of E. coli, by destroying genes encoding translation termination factors and stably introducing tRNA that translates amber codons, and several genes.

This method is applicable not only to E. coli but also to any bacteria, and provides a base for any kind of systems to generate non-natural proteins. By producing modified proteins having more than one non-natural amino acid using our method, novel proteins with novel functions that have not been available so far can be obtained.

A protein is generated in which 6 amber codons are introduced near N-terminal of glutathione-S-transferase (introduction sites are indicated by *). A non-natural amino acid, iodotyrosine, can be incorporated to all sites.

Merits

• More than one non-natural amino acid can be incorporated into non-natural proteins.

Applications

• Production of novel functional proteins having non-natural amino acids

References

• 1.PCT/JP2011/063778
• 2.Mukai T, et al. BBRC. 2011 Aug 12;411(4):757-61.
• 3.Mukai T, et al. Nucleic Acids Res. 2010 Dec;38(22):8188-95.