ABSTRACT

Adding New Chemistry to Proteins via Genetic Incorporation

by

Shuo Chen, B.S., Peking University

Directed by: Professor Meng-Lin Tsao

Professor Tao Ye

Incorporation of unnatural amino acids into proteins provides a powerful way to add new chemistries to proteins, which would be an advantage to study protein behavior, to provide a site-specific protein modification method, and to enhance protein properties. Rather than incorporating unnatural amino acid into proteins by means of chemical methods, recent advances in genetic incorporation of unnatural amino acid into proteins might be an effective way to introduce novel chemical groups into proteins, which shows a promising future in chemical biology studies.

The unnatural amino acid, 2-amino-3- (6-hydroxy-naphthalen-2-yl)propanoic acid (2NpOH) is an analogue of tyrosine, with an orthogonal reactivity and unique fluorescence properties, which can be utilized as site-specific bio-conjugation tag or as a biochemical probe. Based on rational design and random mutation, two aminoacyl-tRNA synthetases (RS-NpOH), which can be utilized to introduce 2-amino-3- (6-hydroxy-naphthalen-2-yl) propanoic acid into protein in vivo in response to the UAG stop codon, were evolved. Protein expression with RS-NpOH showed that the unnatural amino acid could be successfully incorporated into proteins. Both ESI-MS analysis and the strong blue fluorescence acquired by addition of 2NpOH indicated that both aminoacyl-tRNA synthetases had high efficiency and fidelity for selective incorporation of 2NpOH.

A chemoselective azo coupling reaction between the genetically encoded 2-naphthol group and diazotized aniline derivatives was developed. The coupling reaction only required very mild condition of pH 7 at 0 °C, and possessed fast reaction rate, high efficiency and excellent selectivity. Thus, it provides us an alternate method for bio-conjugation, such as PEGylation. Furthermore, to exploit the higher reactivity of the 2-naphthol residue under a nucleophilic addition coupling, an in vivo Mannich type reaction was proposed. It revealed that a cross coupling reaction could achieve between a lysine residue and a 2NpOH residue with the presence of formaldehyde at physiological conditions. Thus, new functionality can be attached to the unnatural amino acid site of proteins, and it would provide us a new tool to study the behavior of proteins and eventually the production of functionally enhanced proteins.

In addition, the fluorescent feature of this unnatural amino acid was also studied upon insertion of the 2NpOH into the GFP chromophore for protein evolution. However, no red-shift fluorescence was observed as expected

In this work, incorporation of two unnatural amino acids into single protein was also demonstrated.

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