Investigation of expression and optimization of recombinant human platelet-derived growth factor

Abstract

Platelet-derived growth factors (PDGFs) are a group of growth factors acting as the primary mitogen for connective tissue cells. Among the different isoforms of the PDGF family, B isoform (i.e. PDGF-B) is of great importance in the body. The role of PDGF-B in wound healing is mediated through chemotactic, mitogenesis and angiogenesis activities in connective tissues. Becaplermin (Regranex)®, is a recombinant form of this growth factor which is used for treatment of diabetic foot ulcers. This protein is a hemodimer with a molecular weight of 24 KDa. Purpose: Investigation of expression and optimization of recombinant human platelet-derived growth factor Material and Methods: In this study, pET-16b vector containing PDGF-B gene was transformed to E.coli cells and protein expression was investigated in different conditions such as temperature, inducer concentration and induction time. Western blotting technique was used for identification of expressed protein. Moreover, pGEX-2T vector was used to study the expression and purification of PDGF-B. Results: Reults of protein expression and purification demonstrated that the expression at temperatures 25 °C and 37 °C was higher than 20 °C and different concentrations of inducers did not significantly alter the protein expression. Moreover, the monomer form of protein with histidine tag was collected with molecular weight of 24 KDa. Additionally, the results of protein purification using GST tag showed that the fusion protein with histidine tag was well purified compared to fusion protein with GST tag. Conclusion: Purification of fusion protein with histidine tag yields better results compared to fusion protein with GST tag in prokaryotic system.