Abstract
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 pPICZA. 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.
Highlights
The yeast Pichia pastoris is used for synthesis of recombinant secretory proteins
coding proteins involved in secretion
of approaches to improve the production of target protein
Summary
Задачей нашего исследования была оценка влияния сверхпродукции протеиндисульфидизомеразы (Pdi) дрожжей P. pastoris на продукцию рекомбинантных интерферонов. Показано влияние суперпродукции Pdi на жизнеспособность и скорость роста клеток, а также на продукцию гетерологичных белков — интерферона-альфа человека и интерферона-гамма курицы. ВЛИЯНИЕ СВЕРХЭКСПРЕССИИ ГЕНА PDI НА ПРОДУКЦИЮ ГЕТЕРОЛОГИЧНЫХ БЕЛКОВ В ДРОЖЖАХ PICHIA PASTORIS. Коэкспрессию гена PDI для увеличения синтеза гетерологичных белков использовали во многих системах экспрессии. Влияние сверхэкспрессии гена PDI на продукцию гетерологичных белков изучали и в дрожжах. Целью нашего исследования было оценить влияние сверхэкспрессии гена PpPDI на уровень продукции модельных гетерологичных белков: интерферона-альфа человека (IFNα16-hum) и интерферона-гамма курицы (IFNγ-chk). Различия в строении белков позволят ответить на вопрос, как влияет сверхэкспрессия гена PpPDI на продукцию интерферонов, и оценить зависимость эффекта от присутствия в белках дисульфидных связей и сайтов гликозилирования
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