Bakteriyel ekspresyon sisteminde safra tuzu hidrolaz (STH) rekombinant protein sekresyonu için ompa sinyal peptidinin test edilmesi

Proteins are allowed to enter the secretory pathway in both prokaryotic and eukaryotic organisms, only if they are accomplished with a specific signal destination which is called a signal peptide (SP). A typical signal peptide consisting of 25-30 residues is located at the N amino-terminal site of proteins and has the task of protein translocation. Mainly the structure of SP has three main parts: the first one is N-region that has the positive-charged domain, the second one is the H-region which is a hydrophobic core, and the third domain is the C-region that is the cleavage site. Signal peptidases are enzymes that bind to the C-site and release the protein in the secretory pathway. The secreted recombinant protein production ability of Escherichia coli is crucial to the biotechnology industry as it minimizes the production cost and downstream processing. However, the large-scale production of recombinant proteins has various obstructions, among which proteolytic degradation and inclusion body formation via proteases are the dominant ones. This dissertation work aims to investigate the secretory effect of the OmpA SP on a pET-based expression system for the production of recombinant bile salt hydrolase (BSH) in E. coli. To demonstrate that the secretory effect of the OmpA signal peptide, the bsh encoding genes and OmpA SP coding gene were cloned into pET-28b(+) plasmid DNA and obtained pEDE3 construct was overexpressed in E. coli BLR(DE3) cells. While the secretory effect of the OmpA SP was observed by the direct plate assay method, the expression levels of the BSH enzyme was detected by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis. Direct plate assay results indicated that pEDE3/BLR(DE3) clones containing OmpA SP and bsh gene secreted BSH enzyme into the medium. However, SDS-PAGE results showed that soluble BSH proteins with 35-42 kDa in size were not visible on a polyacrylamide gel. These results demonstrated that the pEDE3 construct containing OmpA SP had the capacity to produce a releasable active BSH protein, albeit a small amount.