earticle

영어

Recently, Chlorotyrosine containing proteins has the attention of protein engineers for developing novel biomarker as well as in preparing pharmacologically active substances. Similarly, fluorination of recombinant protein is significant for analyzing the structural, biological and physical property of protein. Due to its significance and requirements, in vivo incorporation of Chloro and Fluoro amino acids into the target protein is essential. Recently genetic incorporation of novel functionality embedded within the non canonical amino acids (NCAA) became an indispensable quest to design and manipulate target proteins with new and enhanced properties. The protein modification can be achieved by two different methods 1. "Genetic code engineering" is based on the reassignment of the sense codon through the selective pressure incorporation method (SPI). 2. “ite specific incorporation”methodology use DNA mutagenesis to introduce in-frame termination triplets (e.g. the amber stop codon) that are considered as blank codons for the expansion of the cellular genetic code. In general, genetic code engineering will utilize the host translational machinery (tRNA/tRNA synthetase system), whereas the site specific incorporation requires 21st tRNA/synthetase pair (orthogonal) for NCAA incorporation into protein. Both methods are successfully utilized to incorporate different NCAA into protein for improving its biophysical and functional properties. Recently, we have developed an interesting GFP for NCAA incorporation with improved stability and folding efficiency. Here, the promise of this new GFP is utilized to exemplify the introduction of a halogen group containing aromatic NCAA into Tyr residues of GFP through SPI method. We observed the high level expression of F-Tyr containing GFP (38mg/L), when compared to that of the parent GFP (34mg/L) and medium supplemented with Cl-Tyr (18mg/L). Due to the bulky nature, we expected that Cl-Tyr incorporation might affect the GFP expression level by forming protein aggregates. The dichroic profiles confirmed the overall secondary structural characteristic of the protein remains same even after incorporation of Cl-Tyr and F-Tyr into the protein. The refolding kinetic parameters of chloro variant was almost similar to that of the parent GFP but the fluorine atom incorporation into protein stabilizes protein folding efficiency this result supports the earlier report. As expected the incorporation of halogen group into GFP, alters its spectral properties such as emission maxima and relative fluorescence intensity. Here we report, the successfully incorporation of Cl-Tyr into GFP without altering the host translational machinery. This demonstrates that protein with a NCAA opens new strategies for the design of tailor made proteins with superior to those of the parent protein.