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

Cover Page

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.


Mikhail A Tsygankov

Author for correspondence.
mial.tsygankov@yandex.ru
St Petersburg State University
Russian Federation, Saint Petersburg, Russia

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

Marina V Padkina

mpadkina@mail.ru
St Petersburg State University
Russian Federation, Saint Petersburg, Russia

PhD, leading researcher, Department of Genetics and Biotechnology

  • Гаретова Л.А., Кириенко О.А. Оценка параметров роста микроорганизмов в условиях периодического и непрерывного культивирования. – Хабаровск: Изд-во Тихоокеан. гос. ун-та, 2010. – 16 с. [Garetova LA, Kirienko OA. Ocenka parametrov rosta mikroorganizmov v uslovijah periodicheskogo i nepreryvnogo kul’tivirovanija. Khabarovsk: Izd-vo Tihookean. gos. un-ta; 2010. 16 p. (In Russ.)]
  • Ермилова Е.В., Залуцкая Ж.М., Лапина Т.В., Шишова М.Ф. Количественный анализ экспрессии генов / Под ред. Е.В. Ермиловой. – 2-е изд. – СПб.: ТЕССА, 2011. – 122 c. [Ermilova EV, Zaluckaja ZhM, Lapina TV, Shishova MF. Kolichestvennyj analiz jekspressii genov. Ed by E.V. Ermilovoj. 2nd ed. Saint Petersburg: TESSA; 2011. 122 p. (In Russ.)]
  • Карабельский А.В., Зиновьева Ю.Г., Смирнов М.Н., Падкина М.В. Создание штаммов дрожжей Pichia pastoris — продуцентов химерных белков «альбумин-интерлейкин-2» и «альбумин-интерферон-α16» // Вестник Cанкт-Петербургского университета. – 2009. – Вып. 2. – Сер. 3. – C. 53–63. [Karabel’skij AV, Zinov’eva JuG, Smirnov MN, Padkina MV. Sozdanie shtammov drozhzhej Pichia pastoris producentov himernyh belkov “al’bumin-interlejkin-2” i “al’bumin-interferon-α16”. Vestnik Sankt-Peterburgskogo uni versiteta. 2009;2(3):53-63. (In Russ.)]
  • Леонович О.А., Серкова Н.Н., Рабинович Я.М. Токсичность меркаптоэтанола для различных линий дрожжей Pichia methanolica, растущих на различных источниках углерода // Прикладная биохимия и микробиология. – 2001. – Т. 37. – № 1. – С. 96–99. [Leonovich SA, Serkova NN, Rabinovich IM. Toxicity of mercaptoethanol to mutant strains of the yeast Pichia methanolica growing on different carbon sources. Prikl Biokhim Mikrobiol. 2001;37(1):96-9. (In Russ.)]
  • Маниатис Т., Фрич Э., Сэмбрук Д. Методы генетической инженерии. Молекулярное клонирование / Ред. А.А. Баев, К.Г. Скрябин. – М.: Мир, 1984. – 480 с. [Maniatis T, Fritch EE, Sambrook J. Molecular Cloning: A laboratory manual. New York: Cold Spring Harbor Laboratory Press; 1982:545.]
  • Манн – Уитни, автоматический расчет. Доступно по: http://www.psychol-ok.ru/statistics/mann-whitney/. Ссылка активна на 27.09.2016.
  • Методика выделения ДНК, «Smash and Grab». Доступно по: http://web.mit.edu/biology/guarente/protocols/quickprep.html. Ссылка активна на 01.09.16.
  • Мюльберг А.А. Фолдинг белка. – СПб.: Изд-во С.-Петерб. ун-та, 2004. – 176 с. [Mjul’berg AA. Folding belka. Saint Petersburg: Izd-vo S.-Peterb. un-ta; 2004. 176 p. (In Russ.)]
  • Наровлянский А.Н., Ершов Ф.И, Гинцбург А.Л. Интерфероны: перспективные направления исследований // Иммунология. – 2013. – № 3. – C. 168–172. [Narovljanskij AN, Ershov FI, Gincburg AL. Interferony: perspektivnye napravlenija issledovanij. Immunologija. 2013;3:168-172. (In Russ.)]
  • Падкина М.В., Парфёнова Л.В., Градобоева А.Е., Самбук Е.В. Синтез гетерологичных интерферонов в клетках дрожжей Pichia pastoris // Прикладная биохимия и микробиология. – 2010. – Т. 46. – № 4. – С. 448–455. [Padkina MV, Parfenova LV, Gradoboeva AE, Sambuk EV. Heterologous interferons synthesis in yeast Pichia pastoris. Applied Biochemistry and Microbiology. 2010;46(4):409-414. (In Russ.)]
  • Патент РФ на изобретение № 2380405/ 2007. [Patent RUS No 2380405/ 2007. (In Russ.)]
  • Ребриков Д.В. ПЦР в реальном времени. – 3-е изд. – М.: БИНОМ, 2011. – 223 c. [Rebrikov DV. PCR v real’nom vremeni. Moscow: BINOM; 2011. 223 p. (In Russ.)]
  • Цыганков М.А., Зобнина А.Е., Падкина М.В. Синтез модифицированных, устойчивых к протеолитической деградации интерферонов-гамма в дрожжах Pichia pastoris // Прикладная биохимия и микробиология. – 2014. – Т. 50. – № 4. – С. 429–436. [Tsygankov MA, Zobnina AE, Padkina MV. Synthesis of recombinant gamma interferons resistant to proteolysis in the yeast Pichia pastoris. Applied Biochemistry and Microbiology. 2014;50(4):387-393. (In Russ.)]. doi: 10.7868/S055510991404028X.
  • Шишкин С.С., Еремина Л.С., Ковалев Л.И., Ковалева М.А. AGR2, ERP57/GRP58 и некоторые другие протеин-дисульфидизомеразы человека // Успехи биологической химии. – 2013. – Т. 53. – С. 81–120. [Shishkin SS, Eremina LS, Kovalev LI, Kovaleva MA. AGR2, ERP57/GRP58 i nekotorye drugie protein-disul’fidizomerazy cheloveka. Uspehi biologicheskoj himii. 2013;53:81-120 (In Russ.)]
  • Balamurugan V, Reddy GR, Suryanarayana VV. S. Pichia pastoris: A notable heterologous expression system for the production of foreign proteins — Vaccines. Indian Journal of Biotechnology. 2007;6:175-186.
  • Borth N, Mattanovich D, Kunert R, Katinger H. Effect of increased expression of protein disulfide isomerase and heavy chain binding protein on antibody secretion in a recombinant CHO cell line. Biotechnol Prog. 2005 Jan-Feb;21(1):106-11. doi: 10.1021/bp0498241.
  • Conn PM. The unfolded protein response and cellular stress. Methods Enzymol. 2011;489: xvii. doi: 10.1016/B978-0-12-385116-1.00020-0.
  • Delic M, Valli M, Graf AB, et al. The secretory pathway: exploring yeast diversity. FEMS Microbiol Rev. 2013Nov;37(6):872-914. doi: 10.1111/1574-6976.12020.
  • Farquhar R, Honey N, Murant SJ, et al. Protein disulfide isomerase is essential for viability in Saccharomyces cerevisiae. Gene. 1991;108(1):81-9. doi: 10.1016/0378-1119(91)90490-3.
  • Gasser B, Maurer M, Rautio J, et al. Monitoring of transcriptional regulation in Pichia pastoris under protein production conditions. BMC Genomics. 2007;19;8:179. doi: 10.1186/1471-2164-8-179.
  • Gasser B, Saloheimo M, Rinas U, et al. Protein folding and conformational stress in microbial cells producing recombinant proteins: a host comparative overview. Microbial Cell Factories. 2008;7:11. doi: 10.1186/1475-2859-7-11.
  • Hsu TA, Watson S, Eiden JJ, Betenbaugh MJ. Rescue of immunoglobulins from insolubility is facilitated by PDI in the baculovirus expression system. Protein Expr Purif. 1996 May;7(3):281-8. doi: 10.1006/prep.1996.0040.
  • Humphreys DP, Weir N, Lawson A, et al. Co-expression of human protein disulphide isomerase (PDI) can increase the yield of an antibody Fab’ fragment expressed in Esche richia coli. FEBS Lett. 1996Feb12;380(1-2):194-7. doi: 10.1016/0014-5793(96)00028-2.
  • Kitchin K, Flickinger MC. Alteration of hybridoma viability and antibody secretion in transfectomas with inducible overexpression of protein disulfide isomerase. Biotechnol Prog. 1995Sep-Oct;11(5):565-74. doi: 10.1021/bp00035a011.
  • Liu Z, Liu L, Osterlund T, et al. Improved production of a heterologous amylase in Saccharomyces cerevisiae by inverse metabolic engineering. Appl Environ Microbiol. 2014Sep;80(17):5542-50. doi: 10.1128/AEM.00712-14.
  • Lodi T, Neglia B, Donnini C. Secretion of human serum albumin by Kluyveromyces lactis overexpressing KlPDI1 and KlERO1. Appl Environ Microbiol. 2005Aug;71(8):4359-4363. doi: 10.1128/AEM.71.8.4359-4363.2005.
  • Lowenthal JW, Lambrecht B, van den Berg TP, et al. Avian cytokines — the natural approach to therapeutics. Dev Comp Immunol. 2000Mar-Apr;24(2-3):355-65. doi: 10.1016/s0145-305x(99)00083-x.
  • Macauley-Patrick S, Fazenda ML, McNeil B, Harvey LM. Heterologous protein production using the Pichia pastoris expression system. Yeast. 2005Mar;22(4):249-70. doi: 10.1002/yea.1208.
  • Mattanovich D, Callewaert N, Rouze P, et al. Open access to sequence: browsing the Pichia pastoris genome. Microb Cell Fact. 2009Oct16;8:53. doi: 10.1186/1475-2859-8-53.
  • Mukaiyama H, Tohda H, Takegawa K. Overexpression of protein disulfide isomerases enhances secretion of recombinant human transferrin in Schizosaccharomyces pombe. Appl Microbiol Biotechnol. 2010Apr;86(4): 1135-43. doi: 10.1007/s00253-009-2393-x.
  • Norgaard P, Westphal V, Tachibana C, et al. Functional differences in yeast protein disulfide isomerases. J Cell Biol. 2001Feb5;152(3):553-562. doi: 10.1083/jcb.152.3.553.
  • Ostermeier M, De Sutter K, Georgiou G. Eukaryotic protein disulfide isomerase complements Escherichia coli dsbA mutants and increases the yield of a heterologous secreted protein with disulfide bonds. J Biol Chem. 1996May3;271(18):10616-22.
  • Pichia Technology from RCT. Available from: http://www.pichia.com/science-center/commercialized-products/. Accessed December 1, 2016.
  • Radhakrishnan R, Walter LJ, Hruza A, et al. Zinc mediated dimer of human interferon-alpha 2b revealed by X-ray crystallography. Structure. 1996Dec15;4(12):1453-63. doi: 10.1016/s0969-2126(96)00152-9.
  • The Saccharomyces Genome Database. Available from: http://www.yeastgenome.org/locus/S000000548/overview. Accessed September 1, 2016.
  • Rand JD, Grant CM. The thioredoxin system protects ribosomes against stress-induced aggregation. Mol Biol Cell. 2006Jan;17(1):387-401. doi: 10.1091/mbc.e05-06-0520.
  • Ryabova LA, Desplancq D, Spirin AS, Pluckthun A. Functional antibody production using cell-free translation: effects of protein disulfide isomerase and chaperones. Nat Biotechnol. 1997Jan;15(1):79-84. doi: 10.1038/nbt0197-79.
  • Uniprot No P05015. Available from: http://www.uniprot.org/uniprot/P05015. Accessed October 30, 2016.
  • Uniprot No P49708. Available from: http://www.uniprot.org/uniprot/P49708. Accessed October 30, 2016.
  • Warsame A, Vad R, Kristensen T, Oyen TB. Characterization of a gene encoding a Pichia pastoris protein disulfide isomerase. Biochem Biophys Res Commun. 2001Mar;281(5):1176-82. doi: 10.1006/bbrc.2001.4479.
  • Winter AD, Cormack G, Page AP. Protein disulfide isomerase activity is essential for viability and extracellular matrix formation in the nematode Caenorhabditis elegans. Dev Biol. 2007Aug15;308(2):449-61. doi: 10.1016/j.ydbio.2007.05.041.
  • Wu S, Letchworth GJ. High efficiency transformation by electroporation of Pichia pastoris pretreated with lithium acetate and dithiothreitol. BioTechniques. 2004 Jan;36(1):152-4.
  • Zhang H, He J, Ji Y, et al. The effect of calnexin deletion on the expression level of PDI in Saccharomyces cerevisiae under heat stress conditions. Cell Mol Biol Lett. 2008;13(1):38-48. doi: 10.2478/s11658-007-0033-y.
  • Zhu T, Guo M, Sun C, et al. A systematical investigation on the genetic stability of multi-copy Pichia pastoris strains. Biotechnol Lett. 2009May;31(5):679-84. doi: 10.1007/s10529-009-9917-4.

Supplementary files

Supplementary Files Action
1. Fig. 1. Plasmid construction View (152KB) Indexing metadata
2. 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 View (110KB) Indexing metadata
3. 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 View (22KB) Indexing metadata
4. Fig. 4. Effect of PpPDI gene overexpression on cell growth rate View (24KB) Indexing metadata
5. 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   View (107KB) Indexing metadata

Views

Abstract - 143

PDF (Russian) - 156


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

Creative Commons License
This work is licensed under a Creative Commons Attribution-ShareAlike 4.0 International License.