טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
M.Sc Thesis
M.Sc StudentMalul Elinor
SubjectSite-Directed Labeling of Proteins via a Cell-Free
Translation System
DepartmentDepartment of Biotechnology
Supervisors Professor Asher Schmidt
Professor Yuval Shoham


Abstract

The aim of this research project was to develop an efficient procedure for producing stable-isotope site-directed labeled proteins. When employing this process, a specific residue in a defined position can be labeled. This technique is a powerful tool for structure-function studies of proteins, as it provides the potential to focus on functional regions alone and to achieve direct identification of putative substrate binding residues using solid-state NMR analysis. Additional purpose was to develop an efficient cell-free translation system for large-scale protein production. Several factors were examined for their ability to affect protein production as Escherichia coli strain, culture growth conditions, extract preparation protocols and translation reaction conditions. Once the optimal conditions were determined, the enhanced T7-S30 extract performance was 10-fold higher, resulting in the production of 100 μg active enzyme per milliliter reaction.

The site-specific labeling methodology is based on in-vitro coupling of suppressor tRNA with labeled amino acids and introducing the complex into a cell-free translation system using an amber mutant gene as a template. Three proteins were chosen for site-directed labeling, in which a specific Lysine codon was replaced with a stop (amber) codon: TAG (nonsense mutants). In accordance, a suppressor Lysine-tRNA, with the complementary anticodon sequence was prepared in-vitro. The enzyme required for Lysine-tRNA aminoacylation, lysyl-tRNA synthetase (LysRS) was over expressed and purified using Ni-NTA column chromatography. The recombinant LysRS was used in the aminoacylation reaction, in which the suppressor Lysine-tRNA was charged with the labeled Lysine ([3H] Lys). The selective incorporation of [3H]Lys was examined via nonsense suppression in the presence of the charged [3H]Lys-tRNA. The mutants had no activity, unless the charged tRNA was added, indicating the presence of a site-directed labeled proteins.