Ecological geneticsEcological geneticsЭкологическая генетика1811-09322411-9202Eco-Vector676410.17816/ecogen15221-30ArticlesСтатьиResearch ArticleInfluence of PDI gene overexpression on heterological proteins production in yeast Pichia pastorisВлияние сверхэкспрессии гена PDI на продукцию гетерологичных белков в дрожжах PICHIA PASTORISTsygankovMikhail AЦыганковМихаил Александрович
Russian Federation

engineer-researcher, Laboratory of biochemical genetics, Department of Genetics and Biotechnology

инженер-исследователь, лаборатория биохимической генетики, кафедра генетики и биотехнологии

mial.tsygankov@yandex.ruPadkinaMarina VПадкинаМарина Владимировна
Russian Federation

PhD, leading researcher, Department of Genetics and Biotechnology

д-р биол. наук, ведущий научный сотрудник, лаборатория биохимической генетики, кафедра генетики и биотехнологии

mpadkina@mail.ruSt Petersburg State UniversityФГБУ ВПО «Санкт-Петербургский государственный университет»15062017152VOL 15, NO2 (2017)ТОМ 15, №2 (2017)213003072017Copyright ©; 2017, Tsygankov M.A., Padkina M.V.Copyright ©; 2017, Цыганков М.А., Падкина М.В.2017Tsygankov M.A., Padkina M.V.Цыганков М.А., Падкина М.В.http://creativecommons.org/licenses/by/4.0https://journals.eco-vector.com/ecolgenet/article/view/6764

Summary: Background. The yeast Pichia pastoris is used for synthesis of recombinant secretory proteins. Overexpression of assistant genes, coding proteins involved in secretion, is one of approaches to improve the production of target protein. PpPDI gene encodes P. pastoris yeast protein disulfide isomerase (Pdi). The aim of our study was to evaluate the effect of Pdi overproduction on recombinant interferons (human interferon-alfa16 and chicken interferon-gamma) production.

Materials and Methods. PpPDI gene was cloned under the control of the AOX1 gene promoter in plasmid pPICZαA. Primers for AJ302014.1 nucleotide sequence of NCBI data base were used for PpPDI gene cloning. The chromosomal DNA of the GS115 strain was used as a template. To generate strains with PpPDI gene overexpression we used a previously obtained strains producing human interferon-alfa16 and chicken interferon-gamma. Yeast transformation was performed by electroporation. Cultivation was performed using single and two-stage strategies in standard media containing methanol as the sole carbon source to induce the AOX1 gene promoter.

Results. We obtained interferon-producing strains with PpPDI gene overexpression. Over-expression of the PpPDI gene in yeast P. pastoris increases the production of interferon-alfa16, a protein containing disulfide bonds, regardless of the mode of cultivation. Effect of PpPDI gene over-expression on the production of interferon-gamma – the protein without disulfide bonds, depends on cultivation mode.

Conclusion. PpPDI gene overexpression can be used to enhance the production of interferons and other proteins that contain disulfide bonds. Effect of PpPDI gene overexpression on recombinant proteins without disulfide bonds may depend on cultivation procedure.

Дрожжи Pichia pastoris используются для синтеза секреторных рекомбинантных белков. Одним из подходов к повышению продукции целевых белков является сверхэкспрессия генов-помощников, белковые продукты которых участвуют в процессе секреции. Задачей нашего исследования была оценка влияния сверхпродукции протеиндисульфидизомеразы (Pdi) дрожжей P. pastoris на продукцию рекомбинантных интерферонов. В ходе нашей работы получены штаммы дрожжей P. pastoris, экспрессирующие ген PpPDI под контролем промотора алкогольоксидазы-1 (AOX1). Показано влияние суперпродукции Pdi на жизнеспособность и скорость роста клеток, а также на продукцию гетерологичных белков - интерферона-альфа16 человека и интерферона-гамма курицы.

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