Influence of PDI gene overexpression on heterological proteins production in yeast Pichia pastoris

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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 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.

About the authors

Mikhail A Tsygankov

St Petersburg State University

Author for correspondence.
Email: mial.tsygankov@yandex.ru

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

Russian Federation, Saint Petersburg, Russia

Marina V Padkina

St Petersburg State University

Email: mpadkina@mail.ru

PhD, leading researcher, Department of Genetics and Biotechnology

Russian Federation, Saint Petersburg, Russia

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Supplementary files

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2. Fig. 1. Plasmid construction

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3. Fig. 2. SDS-PAGE analysis of proteins from culture fluid of productive strains with PpPDI gene overexpression: M1, M2 – protein markers of a certain molecular weight (kDa), (K- ,1,2,3,4) – protein components from culture fluid of these strains: [K-] – GS115, [1] – GS-humIFNA16, [2] – GS-humIFNA16-AOX1PDI, [3] – GS-chkIFNG, [4] – GS-chkIFNG-AOX1PDI

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4. Fig. 3. PpPDI gene copy number determination in the obtained strains: Strains: [1] – GS115, [2] – GS-humIFNA16-AOX1PDI, [3] – GS-chkIFNG-AOX1PDI, [4] – GS-HIS, [5] – GS-AOX1PDI

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5. Fig. 4. Effect of PpPDI gene overexpression on cell growth rate

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6. Fig. 5. Western-blot analysis of proteins from culture fluids: [M] – protein marker of a certain molecular weight (kDa). Culture fluid probes of strains: [K-] – GS-aox1 in one step cultivation, [1] – GS-humIFNA16 in one step cultivation and [2] – in two step cultivation  

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Copyright (c) 2017 Tsygankov M.A., Padkina M.V.

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