Cloning and expression of ocriplasmin in Pichia pastoris expression system

Ocriplasmin, a 27 kDa serine protease, represents a new treatment option for numerous vitreoretinopathies involving symptomatic vitreomacular adhesion (VMA) and vitreomacular traction. When given as an intravitreal injection, ocriplasmin degrades fibronectin and laminin, results in posterior vitreous detachment (PVD). The methylotrophic yeast Pichia. Pastoris is a highly efficient expression system that is used in molecular biology for production of recombinant proteins. Being a eukaryote, P.pastoris is capable of doing many of the post-translational modifications performed by higher eukaryotic cells including proteolytic processing, proper folding, disulfidebond formation and glycosylation. The aim of this study was to evaluate the expression of recombinant Ocriplasmin in P. pastoris expression system. Ocriplasmin gene was cloned into a high copy number vector, pPink-Hc and transferred first to E. coli Top10. PichiaPink™ vector containing the ocriplasmin was linearized by cutting at a unique site to promote integration into the P. pastoris genome and transformed into P. pastoris competent cells. The transformed cells were selected on MD agar selection plates after incubation at 28°C for 6 days. A P. pastoris clone containing Ocriplasmin was cultured in BMGY and the expression was carried out using BMMY medium and analyzed by SDS-PAGE. Screening of transformed cells on MD agar results in several colonies that were positive for the target gene. Expression of recombinant Ocriplasmin in P. pastoris results in 27 kDa protein that was in expected size. The result of this study indicated that P. pastoris is an efficient expression system for production of recombinant Ocriplasmin

کلیدواژه ها:

Ocriplasmin ، Pichia pastoris ، Cloning ، Expression