Cell-Free Incorporation of Unnatural Photo-Cross Linkable Amino Acid p-Benzoyl-L-Phenylalanine (pBpa) Into γ-Secretase Subunit (Presenilin-1 (PS1)) رسالة ماجسير

Abstract

The γ-secretase (GS) complex is critical for regulating the processing of amyloid precursor protein (APP), which is intricately associated with the development of Alzheimer's disease (AD). The presenilin-1 (PS1) subunit, central of GS, contains the catalytic core necessary GS's proteolytic activity. Understanding PS1's structural and functional dynamics within the GS is crucial for elucidating PS1's role in AD. Recent developments in protein engineering, especially the site-specific incorporation of unnatural amino acids (UAAs) like p-Benzoyl-L-phenylalanine (pBpa) through genetic code expansion, have improved the capacity to examine these dynamics. pBpa, can form covalent bonds with nearby residues when activated by ultraviolet (UV) light making it essential for investigating protein interactions and conformational changes. This study aims to develop a Cell-Free Protein Synthesis (CFPS) system to incorporate the pBpa at the 190th amino acid position in PS1 using an amber stop codon and an orthogonal aminoacyl-tRNA synthetase (aaRS)/tRNA pair within an E. coli lysate-based CFPS system. This includes designing and optimizing of DNA templates, preparing of the required components, and refining of the CFPS system for efficient protein labeling and expression. Methanococcus jannaschii tyrosyl-tRNA (tRNACUAopt) was produced in BL21 Star™ (DE3) and isolated using the phenol-chloroform extraction method. Modified Methanococcus jannaschii tyrosyl-tRNA synthetase (Bpa-RS) was expressed in BL21γ::DE3 and purified using immobilized metal affinity chromatography (IMAC). The CFPS system was optimized using enhanced green fluorescent protein (eGFP) as a reporter. Agarose gel electrophoresis, sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and western blot were used for analysis. Key plasmids and proteins for CFPS system were successfully verified. Double digestion and electrophoresis verified the existence of the Bpa-RS, tRNACUAopt, PS1-N190-amber, and GFP genes. Bpa-RS and TEV protease were successfully expressed and purified with the correct size bands verified by SDS-PAGE and western blot. The optimal volume ratio of Bpa-RS to TEV protease was established for effective His-tag cleavage. tRNA was effectively expressed and isolated. The truncated PS1 version resulted from the CFPS system, indicating a need for further optimization to incorporate pBpa efficiently.

Key aspects of the CFPS system for PS1 protein synthesis, including plasmid verification, expression and purification of Bpa-RS, tRNA isolation, and plasmid amount optimization, were successfully addressed in this study. However, further optimization is required in order to yield full-length PS1. The outcomes underscore CFPS's potential in protein synthesis and its applications in drug discovery, industrial biotechnology, and synthetic biology, especially in developing treatments for diseases like AD. This study was conducted in the laboratories of the Centre for Structural Systems Biology (CSSB) in Hamburg, Germany, from October 2021 to June 2022, with funding from the Palestinian-German Science Bridge (PGSB).