Obtaining, purification and characterization of soluble form of prolyl endopeptidaze (PEP) enzyme in Escherichia coli expression system

Prolyl endopeptidase is a large cytosolic enzyme belonging to the serine peptidase class and has been isolated from Aspergillus niger, a frequently cultured microorganism. Prolyl endopeptidase is a promising therapeutic agent due to its ability to degrade proline-rich prolamines and inhibits their inflammatory reaction. Today, it is thought that oral administration of prolyl endopeptidase enzyme will be an effective strategy in the treatment of celiac disease. The purpose of this treatment is to prevent the breakdown of prolyl endopeptidase by stomach acids and to ensure that it is effective in the small intestine. Problems occur during the recombinant production of the targeted prolyl endopeptidase enzyme in Escherichia coli. Inclusion bodies are formed during high level expression of prolyl endopeptidase in Escherichia coli. Strategies used to recover protein from inclusion bodies that occur during recombinant protein production may take a long time and contain various difficulties in practice. In the scope of this thesis, Escherichia coli BL21 (DE3) cells were used for the expression of prolyl endepeptidase enzyme. By using five chaperone plasmid systems. (pG-KJE8, pGro7, pKJE7, pG-Tf2, pTf16), prolyl endopeptidase enzyme was prevented from forming inclusion bodies and its production in soluble form was tested. As a result of the experiments, it was observed that plasmid pKJE7 helped the production of prolyl endopeptidase enzyme in some soluble form. Activity studies of the prolyl endopeptidase were carried out using spectrophotometric analysis and the Km value of prolyl endopeptidase enzyme was found to be 2.06 mM and Vm= 0.0359 mM / sec.