Expression, purification and quantification study of revised modified NhKRS

Abstract

By Ha Nguyen, Biochemistry

Advisor: Pearl Tsang

Presentation ID: PM_D03

Abstract: Lysyl-tRNA synthetase (LysRS) is an enzyme that is essential for protein formation because it is responsible for correct charging of lysyl tRNA with the amino acid lysine. In prokaryotic cells, the enzyme consists of two domains- the binding domain for the anticodon of the tRNA for proper recognition, and the catalytic domain at the C-terminus. Eukaryotic LysRS contains an additional N-terminal domain (NTD). The overall function of this N-terminal is not well understood. There have been studies that show that this N-terminal extension increases HIV-1's selective incorporation of tRNALys. tRNALys has been shown to be used as a primer for reverse transcriptase of HIV-1. As LysRS is incorporated, it is accompanied by tRNALys and this process is essential to the HIV-1 life cycle. Based on this evidence, the focus of this research is to describe approaches that are suitable for gene expression of N-terminal domain in Escherichia coli (BL21 DE3 strain) and purification of a specific protein-RMN, revised modified NTD protein. Moreover, a quantification study was performed to determine the concentration of the purified protein using spot densitometry. Due to its sequence, RMN protein is difficult to quantify by UV-spectrophotometry. Western blot technique is also used to test for the presence of His-tag to determine how well the protein was purified. After the protein was purified, the second goal for this research would be to investigate the structure and the affinity binding of the protein to RNA oligonucleotides that mimic portions of the full tRNALys.