Construction of a Producer Strain of the Fibrinolitic Enzyme PAPC Based on Pichia pastoris Yeast

Abstract— Objective: Yeast Pichia pastoris (Komagataella phaffii) is widely used in food and pharmaceutical industries as microbial cell factories of recombinant proteins. Protease-activator of protein C (PAPC) of blood plasma from Aspergillus ochraceus VKM F-4104D micromycetes can potentially be introduced into therapeutic practice as a fibrinolytic drug and into diagnostic systems for blood coagulation analysis as the main component that activates protein C. The aim of this work was to construct a PAPC-producing strain based on P. pastoris and to demonstrate the effective production and secretion of the recombinant enzyme into the culture medium. Methods: We assembled a pD912-AFSnoEAEA-PAPC vector carrying the papc gene under a methanol-induced AOX1-promoter. This vector was used to transform P. pastoris yeast, and transformants were selected on a zeocin-containing medium. The clones most effectively producing the target enzyme were selected using an agar medium with casein and an analysis of the culture medium by SDS-PAGE. Results and Discussion: The selected clones secreted an active form of PAPC into the culture medium. The ability to production of the target enzyme remained after 60 generations. LC-MS analysis confirmed the presence of the enzyme in the culture medium and demonstrated that accumulation occurs in the mature active form. Conclusions: We demonstrated that P. pastoris system can be successfully used for the high-level expression of PAPC from A. ochraceus. The low amount of host cell proteins in the culture medium makes the subsequent purification of the target enzyme easier in comparison to bacterial expression systems. The obtained strain can be used for the further production of experimental industrial batches of the enzyme in biotechnological production facilities that support yeast fermentation.