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

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Yeast Pichia pastoris (Komagataella phaffii) is widely used in food and pharmaceutical industries as microbial cell factories of recombinant proteins due to its ability to heterologously overexpress many target proteins, including proteolytic enzymes. Protease-activator of protein C (PAPC) of blood plasma from micromycete Aspergillus ochraceus VKM F-4104D 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. To solve problems of using protease-activator of protein C in medicine and veterinary science, it is important to have a reliable system to produce the recombinant enzyme. Such a production system can be created on the basis of yeast. The aim of this work was to construct a PAPC-producing strain based on P. pastoris and to demonstrate effective production and secretion of the recombinant enzyme into the culture fluid. We assembled a vector carrying the papc gene. 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 agar medium with casein and analysis of the culture fluid by SDS-PAGE. The dynamics of accumulation of the active form of PAPC in the culture fluid after induction of protein synthesis during submerged cultivation of the producing clone in a flask was studied. LC-MS analysis confirmed the presence of the enzyme in the culture medium and demonstrated that accumulation occurs in the mature active form. The obtained strain can be used for further production of experimental industrial batches of the enzyme in biotechnological production facilities that support yeast fermentation.

作者简介

S. Komarevtsev

Shenyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences

Moscow, Russia

Yu. Lapteva

Pushchino Scientific Center for Biological Research of the Russian Academy of Sciences, Institute for Biological Instrumentation

Pushchino, Russia

R. Ziganshin

Shenyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences

Moscow, Russia

V. Farofonova

Pushchino Scientific Center for Biological Research of the Russian Academy of Sciences, Institute for Biological Instrumentation

Pushchino, Russia

V. Stepanenko

Sechenov First Moscow State Medical University

Moscow, Russia

A. Osmolovsky

Lomonosov Moscow State University, Faculty of Biology

Moscow, Russia

K. Miroshnikov

Shenyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences; Lomonosov Moscow State University, Faculty of Biology

Moscow, Russia; Moscow, Russia

E. Loktyushov

Pushchino Scientific Center for Biological Research of the Russian Academy of Sciences, Institute for Biological Instrumentation

Email: zhenyaloktushov@gmail.com
Pushchino, Russia

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