The Highly Efficient Expression System of Recombinant Human Prolidase and the Effect of N-Terminal His-Tag on the Enzyme Activity

• Published in: Cells (Basel, Switzerland) Vol. 11; no. 20; p. 3284
• Main Authors: Czyrko-Horczak, Justyna, Nizioł, Magdalena, Forlino, Antonella, Besio, Roberta, Miltyk, Wojciech
• Format: Journal Article
• Language: English
• Published: Basel MDPI AG 01.10.2022; MDPI
• Subjects: Affinity chromatography; Binding sites; Biological activity; C-Terminus; Cellular signal transduction; Collagen; E coli; Enzymatic activity; Enzymes; Gene expression; Genetic aspects; Glycerol; High temperature; human recombinant prolidase; Physiological aspects; Prolidase; prolidase activity; protein expression system; Proteins; Poland; Canada; United States > US
• ISSN: 2073-4409; 2073-4409
• DOI: 10.3390/cells11203284

Prolidase is an enzyme hydrolyzing dipeptides containing proline or hydroxyprolineat the C-terminus and plays an important role in collagen turnover. Human prolidase is active as a dimer with the C-terminal domain containing two Mn2+ ions in its active site. The study aimed to develop a highly efficient expression system of recombinant human prolidase (rhPEPD) and to evaluate the effect of the N-terminal His-Tag on its enzymatic and biological activity. An optimized bacterial expression system and an optimized purification procedure for rhPEPD included the two-step rhPEPD purification procedure based on (i) affinity chromatography on an Ni2+ ion-bound chromatography column and (ii) gel filtration with the possibility of tag removal by selective digestion with protease Xa. As the study showed, a high concentration of IPTGand high temperature of induction led to a fast stimulation of gene expression, which as a result forced the host into an intensive and fast production of rhPEPD. The results demonstrated that a slow induction of gene expression (low concentration of inducing factor, temperature, and longer induction time) led to efficient protein production in the soluble fraction. Moreover, the study proved that the presence of His-Tag changed neither the expression pattern of EGFR-downstream signaling proteins nor the prolidase catalytic activity.