vavilovВавиловский журнал генетики и селекцииVavilov Journal of Genetics and Breeding2500-3259Institute of Cytology and Genetics of Siberian Branch of the RAS10.18699/VJ21.102vavilov-3207Research ArticleАКТУАЛЬНЫЕ ТЕХНОЛОГИИMAINSTREAM TECHNOLOGIESПолучение рекомбинантного штамма Komagataella phaffi – продуцента протеиназы К из Tritirachium albumCreation of a recombinant Komagataella phaffii strain, a producer of proteinase K from Tritirachium albumhttps://orcid.org/0000-0001-9732-1246БеклемишевА. Б.BeklemishevA. B.

Новосибирск, Россия

Novosibirsk, Russia

beklem@niibch.ruhttps://orcid.org/0000-0002-7808-9274ПыхтинаМ. Б.PykhtinaM. B.

Новосибирск, Россия

Novosibirsk, Russia

https://orcid.org/0000-0001-6916-6875КуликовЯ. М.KulikovYa. M.

Новосибирск, Россия

Novosibirsk, Russia

https://orcid.org/0000-0002-7445-0608ГорячковскаяТ. Н.GoryachkovskayaT. N.

Новосибирск, Россия

Novosibirsk, Russia

https://orcid.org/0000-0002-0764-5800БочковД. В.BochkovD. V.

Новосибирск, Россия

Novosibirsk, Russia

СергееваС. В.SergeevaS. V.

Новосибирск, Россия

Novosibirsk, Russia

ВасильеваА. Р.VasilevaA. R.

Новосибирск, Россия

Novosibirsk, Russia

РомановВ. П.RomanovV. P.

Новосибирск, Россия

Novosibirsk, Russia

НовиковаД. С.NovikovaD. S.

г. Алексеевка, Белгородская область, Россия

Alekseyevka, Belgorod region, Russia

https://orcid.org/0000-0002-7122-2546ПельтекС. Е.PeltekS. E.

Новосибирск, Россия

Novosibirsk, Russia

beklem@niibch.ruФедеральный исследовательский центр фундаментальной и трансляционной медицины; Федеральный исследовательский центр Институт цитологии и генетики Сибирского отделения Российской академии наукРоссияFederal Research Center of Fundamental and Translational Medicine; Institute of Cytology and Genetics of the Siberian Branch of the Russian Academy of SciencesRussian FederationФедеральный исследовательский центр Институт цитологии и генетики Сибирского отделения Российской академии наукРоссияInstitute of Cytology and Genetics of the Siberian Branch of the Russian Academy of SciencesRussian FederationАкционерное общество «Эфирное», г. АлексеевкаРоссияEfirnoe Joint-Stock CompanyRussian Federation202101012022258882888Copyright © Беклемишев А.Б., Пыхтина М.Б., Куликов Я.М., Горячковская Т.Н., Бочков Д.В., Сергеева С.В., Васильева А.Р., Романов В.П., Новикова Д.С., Пельтек С.Е., 20222022Беклемишев А.Б., Пыхтина М.Б., Куликов Я.М., Горячковская Т.Н., Бочков Д.В., Сергеева С.В., Васильева А.Р., Романов В.П., Новикова Д.С., Пельтек С.Е.Beklemishev A.B., Pykhtina M.B., Kulikov Y.M., Goryachkovskaya T.N., Bochkov D.V., Sergeeva S.V., Vasileva A.R., Romanov V.P., Novikova D.S., Peltek S.E.This work is licensed under a Creative Commons Attribution 4.0 License.https://vavilov.elpub.ru/jour/article/view/3207

Объектами исследования являлись рекомбинантные штаммы Komagataella phafi K51, несущие интегрированный в их геном гетерологичный ген протеиназы К (PK-w) из Tritirachium album, а также препарат рекомбинантной протеиназы К, полученный из этих штаммов. Целью работы было изучение возможности получения рекомбинантных штаммов K. phafi K51, обеспечивающих высокий уровень синтеза функционально активной протеиназы К из T. album, и анализ ферментативной активности полученного рекомбинантного энзима. В работе использованы методы компьютерного анализа первичной структуры гена протеиназы К, молекулярно-биологические методы (ПЦР, электрофорез ДНК в агарозных гелях, электрофорез белков в SDS-ПААГ в денатурирующих условиях, спектрофотометрия, методы количественного определения активности протеаз), генно-инженерные методы (методы клонирования и селекции генов в бактериальных клетках Escherichia coli str. TOP10 и в метилотрофных дрожжах K. phafi str. K51). Спроектирован ген природной протеиназы К (PK-w), оптимизированный для экспрессии в дрожжах K. phafi K51. Осуществлены синтез и клонирование синтезированного гена протеиназы К в составе вектора pPICZα-A в клетках E. coli str. TOP10. Ген протеиназы К встроен в векторную плазмиду pPICZα-A таким образом, чтобы на последующем этапе переклонирования в клетках дрожжей обеспечить его эффективную экспрессию под контролем промотора и терминатора гена AOX1, а продукт экспрессии клонированного гена содержал сигнальный пептид альфа-фактора Saccharomyces cerevisiae для обеспечения секреции белка в культуральную жидкость. Проведено переклонирование рекомбинантной плазмиды (pPICZα-A/PK-w) в клетках дрожжей K. phafi str. K51. Получен рекомбинантный штамм K. phafi K51, несущий синтетический ген протеиназы К и обеспечивающий его экспрессию в дрожжах и секрецию в культуральную среду. Приблизительный выход рекомбинантной протеиназы К после четырех суток культивирования дрожжевых рекомбинантных клонов составил 25 мкг/мл. Полученный препарат рекомбинантной протеазы обладает высокой удельной протеолитической активностью, составляющей ~5000 Ед/мг.

The objects of the study were recombinant clones of Komagataella phaffii K51 carrying the heterologous proteinase K (PK-w) gene from Tritirachium album integrated into their genome as well as samples of recombinant proteinase K isolated from these clones. The aims of this work were i) to determine whether it is possible to create recombinant K. phaffii K51 clones overexpressing functionally active proteinase K from T. album and ii) to analyze the enzymatic activity of the resulting recombinant enzyme. The following methods were used: computational analysis of primary structure of the proteinase K gene, molecular biological methods (PCR, electrophoresis of DNA in an agarose gel, electrophoresis of proteins in an SDS polyacrylamide gel under denaturing conditions, spectrophotometry, and quantitative assays of protease activity), and genetic engineering techniques (cloning and selection of genes in bacterial cells Escherichia coli TOP10 and in the methylotrophic yeast K. phaffii K51). The gene encoding natural proteinase K (PK-w) was designed and optimized for expression in K. phaffii K51. The proteinase K gene was synthesized and cloned within the plasmid pPICZα-A vector in E. coli TOP10 cells. The proteinase K gene was inserted into pPICZα-A in such a way that – at a subsequent stage of transfection into yeast cells – it was efficiently expressed under the control of the promoter and terminator of the AOX1 gene, and the product of the exogenous gene contained the signal peptide of the Saccharomyces cerevisiae a-factor to ensure the protein’s secretion into the culture medium. The resultant recombinant plasmid (pPICZα-A/PK-w) was transfected into K. phaffii K51 cells. A recombinant K. phaffii K51 clone was obtained that carried the synthetic proteinase K gene and ensured its effective expression and secretion into the culture medium. An approximate productivity of the yeast recombinant clones for recombinant proteinase K was 25 μg/ mL after 4 days of cultivation. The resulting recombinant protease has a high specific proteolytic activity: ~5000 U/mg.

протеиназа Кклонирование генаKomagataella phafiэкспрессия генаактивность ферментаproteinase Kgene cloningKomagataella phaffiigene expressionenzymatic activityThis work was done with financial support from the Ministry of Science and Higher Education of the Russian Federation as part of the implementation of a comprehensive project on the topic “Creation of a high-tech production workflow for high-quality nutritional proteins of plant origin” (in accordance with the agreement No. 075-11-2020-036 of December 15, 2020, about a subsidy from the federal budget for the development of cooperation between a state scientific institution and an enterprise from a real-world sector of the economy in order to implement a comprehensive project for setting up high-tech production) at the ICG SB RAS within the framework of Decree No. 218 of the Russian Government (April 9, 2010). 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The authors declare that there are no conflicts of interest present.