Biography & Net Worth: Team Engineer Instructions-Evolution of Translation Systems for Efficient Unnatural Amino Acid Incorporation

Amino acid selenocysteine, 3d ball model. credit: YassineMrabet / CC BY 3.0 / Wikipedia

The genetic code of all three kingdoms of life is universal and encodes the same 20 natural amino acids for various complex physiological functions. The expansion of the genetic code by including universal amino acids (UAAs) with diverse functional groups has enabled the synthesis of advanced or novel proteins and the creation of UAA-dependent synthetic auxotrophy. Rice fields.

However, when UAA is used in place of natural amino acids, it is very difficult to construct an in vivo site-specific UAA uptake system with comparable efficiency and similar protein yields. In 2020, Lin Shixian et al. We have reported the design of a widely used orthogonal chimeric translation system that operates in bacterial and mammalian systems for post-translational modification and expansion of the fluorescent UAA genetic code. However, like all existing orthogonal translation systems, the efficiency of the chimeric system is much lower than that of the endogenous translation system of natural amino acids, which hinders its application for protein design and functional studies.

On December 2, a team led by Professor Lin Shixian from the Institute of Life Sciences, Zhejiang University, published a report titled “Directed Development of Translational Systems for Efficient Unnatural Amino Acid Uptake and Generalized Synthetic Nutritional Requirements”. The article has been published. Creation “in the magazine” nature communication.. In this paper, the researchers report the directed evolution of a translational system that allows the incorporation of unnatural amino acids with the same efficiency as natural amino acids and the creation of synthetic auxotrophy in a general manner. ,

In this study, Lin and his team achieved wild-type efficiency in the production of full-length proteins, and improved uptake efficiency of chimeric phenylalanine (PHE), with low background activity in the absence of UA. I designed the system. These desirable properties have been demonstrated by the directed evolution of chimeric Phe tRNA (chPheT) and chimeric Phe synthetase (chPheRS) in the acceptor stem into several Phe and tryptophan (Trp) analogs.

After screening ~1.7×10 libraries7 With a tRNA molecule and 13,000 synthetase clones, the researchers have obtained mutants that can significantly improve the efficiency of the chimeric translation system in UAA recognition. The chimera that evolved with the direction translation system was the model protein and the multiple sites of many functional proteins were able to encode multiple UAAs. Efficiently as natural. amino acids.. Meanwhile, it showed significantly higher fidelity. Impressively, the designed chPheRS/chPheT pair showed a 65-fold increase in amber suppression efficiency. Subsequently, a chPheRS/chPheT pair engineered to incorporate Azf was introduced into a commercially available E. coli strain to construct a UAA-dependent synthetic auxotrophic strain.

“By introducing an in-frame amber codon into an essential gene, we were able to identify some synthetic auxotrophic strains that are strictly dependent on externally supplied UAA for growth,” Lin said. says Mr. “This study provides a powerful and general technique for the synthesis of UAA-dependent synthetic auxotrophic vaccines for the prevention of genetically modified organisms and the development of live attenuated vaccines,” he said.

Expand the genetic code with the set of four codons

for more information:
Hongxia Zhao et al, Directed development of translation systems for efficient unnatural amino acid uptake and generalized synthetic auxotrophy construction, nature communication (2021). DOI: 10.1038/s41467-021-27399-x

Provided by Zhejiang University

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Team Engineers Development of Translation System for Instruction-Efficient Unnatural Amino Acid Incorporation

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