Comparasion of the effectiveness of anchor proteins ScAGα1p, KpCW51p, KpCW61p for surface display in yeast Komagataella phaffii

Abstract

BACKGROUND: Yeast display is an effective technology for exposure target proteins to the cell surface by fusing them with cell wall proteins. This technique, among other things, makes it possible to obtain vaccine preparations based on yeast by exposing antigen proteins on their cell surface. Finding and selecting proteins that allow effective exposure of target proteins on the surface of yeast cells is an urgent task.

AIM: The aim of this work was to evaluate the efficiency of cell wall proteins ScAGα1p, KpCW51p, KpCW61p for displaying the reporter protein on the Komagataella phaffii cell surface, including the study of several variants of the ScAGα1 gene coding sequence.

MATERIALS AND METHODS: The studied gene sequences were cloned under the control of the AOX1 gene promoter in the same reading frame as the eGFP reporter protein gene and integrated into the genome of the K. Phaffii yeast strain X-33.

RESULTS: Cytoimmunochemical analysis and confocal microscopy of strains displaying the eGFP protein on their surface under conditions of induction of the AOX1 gene promoter made it possible to identify the most effective anchor protein. The best efficiency was demonstrated for the sequence of the ScAGα1 gene without the native 3' non-coding region.

CONCLUSIONS: The plasmids obtained in the work will make it possible to obtain a yeast strain K. phaffii that effectively exposure proteins, including antigens, on its surface, which can be used as a vaccine preparation.

Keywords

yeast display / Pichia pastoris / Komagataella phaffii / heterologous protein synthesis

References

[1] Smith GP. Filamentous fusion phage: novel expression vectors that display cloned antigens on the virion surface. Science. 1985;228(4705):1315–1317. DOI: 10.1126/science.4001944

[2] Smith G.P. Filamentous fusion phage: novel expression vectors that display cloned antigens on the virion surface // Science. 1985. Vol. 228, No. 4705. P. 1315–1317. DOI: 10.1126/science.4001944

[3] Ueda M. Establishment of cell surface engineering and its development. Biosci Biotechnol Biochem. 2016;80(7):1243–1253. DOI: 10.1080/09168451.2016.1153953

[4] Ueda M. Establishment of cell surface engineering and its development // Biosci Biotechnol Biochem. 2016. Vol. 80, No. 7. P. 1243–1253. DOI: 10.1080/09168451.2016.1153953

[5] Schreuder MP, Brekelmans S, van den Ende H, Klis FM. Targeting of a heterologous protein to the cell wall of Saccharomyces cerevisiae. Yeast. 1993;9(4):399–409. DOI: 10.1002/yea.320090410

[6] Schreuder M.P., Brekelmans S., van den Ende H., Klis F.M. Targeting of a heterologous protein to the cell wall of Saccharomyces cerevisiae // Yeast. 1993. Vol. 9, No. 4. P. 399–409. DOI: 10.1002/yea.320090410

[7] Boder ET, Wittrup KD. Yeast surface display for screening combinatorial polypeptide libraries. Nat Biotechnol. 1997;15(6):553–557. DOI: 10.1038/nbt0697-553

[8] Boder E.T., Wittrup K.D. Yeast surface display for screening combinatorial polypeptide libraries // Nat Biotechnol. 1997. Vol. 15, No. 6. P. 553–557. DOI: 10.1038/nbt0697-553

[9] Kurtzman CP. Biotechnological strains of Komagataella (Pichia) pastoris are Komagataella phaffii as determined from multigene sequence analysis. J Ind Microbiol Biotechnol. 2009;36(11):1435–1438. DOI: 10.1007/s10295-009-0638-4

[10] Kurtzman C.P. Biotechnological strains of Komagataella (Pichia) pastoris are Komagataella phaffii as determined from multigene sequence analysis // J Ind Microbiol Biotechnol. 2009. Vol. 36, No. 11. P. 1435–1438. DOI: 10.1007/s10295-009-0638-4

[11] Kim S-Y, Sohn J-H, Pyun Y-R, Choi E-S. A cell surface display system using novel GPI-anchored proteins in Hansenula polymorpha. Yeast. 2002;19(13):1153–1163. DOI: 10.1002/yea.911

[12] Kim S.-Y., Sohn J.-H., Pyun Y.-R., Choi E.-S. A cell surface display system using novel GPI-anchored proteins in Hansenula polymorpha // Yeast. 2002. Vol. 19, No. 13. P. 1153–1163. DOI: 10.1002/yea.911

[13] Tanaka T, Yamada R, Ogino C, Kondo A. Recent developments in yeast cell surface display toward extended applications in biotechnology. Appl Microbiol Biotechnol. 2012;95(3):577–591. DOI: 10.1007/s00253-012-4175-0

[14] Tanaka T., Yamada R., Ogino C., Kondo A. Recent developments in yeast cell surface display toward extended applications in biotechnology // Appl Microbiol Biotechnol. 2012. Vol. 95, No. 3. P. 577–591. DOI: 10.1007/s00253-012-4175-0

[15] Bielen A, Tepariс R, Vujaklija D, Mrsa V. Microbial anchoring systems for cell-surface display of lipolytic enzymes. Food Technol Biotechnol. 2014;52(1):16–34.

[16] Bielen A., Tepariс R., Vujaklija D., Mrsa V. Microbial anchoring systems for cell-surface display of lipolytic enzymes // Food Technol Biotechnol. 2014. Vol. 52, No. 1. P. 16–34.

[17] Wasilenko JL, Sarmento L, Spatz S, Pantin-Jackwood M. Cell surface display of highly pathogenic avian influenza virus hemagglutinin on the surface of Pichia pastoris cells using alpha-agglutinin for production of oral vaccines. Biotechnol Prog. 2010;26(2):542–547. DOI: 10.1002/btpr.343

[18] Wasilenko J.L., Sarmento L., Spatz S., Pantin-Jackwood M. Cell surface display of highly pathogenic avian influenza virus hemagglutinin on the surface of Pichia pastoris cells using alpha-agglutinin for production of oral vaccines // Biotechnol Prog. 2010. Vol. 26, No. 2. P. 542–547. DOI: 10.1002/btpr.343

[19] Lei H, Jin S, Karlsson E, et al. Yeast surface-displayed H5N1 avian influenza vaccines. J Immunol Res. 2016;2016:4131324. DOI: 10.1155/2016/4131324

[20] Lei H., Jin S., Karlsson E., et al. Yeast surface-displayed H5N1 avian influenza vaccines // J Immunol Res. 2016. Vol. 2016. ID4131324. DOI: 10.1155/2016/4131324

[21] Angelini A, Chen TF, de Picciotto S, et al. Protein engineering and selection using yeast surface display. Methods Mol Biol. 2015;1319:3–36. DOI: 10.1007/978-1-4939-2748-7_1

[22] Angelini A., Chen T.F., de Picciotto S., et al. Protein Engineering and selection using yeast surface display // Methods Mol Biol. 2015. Vol. 1319. P. 3–36. DOI: 10.1007/978-1-4939-2748-7_1

[23] Cherf GM, Cochran JR. Applications of yeast surface display for protein engineering. Methods Mol Biol. 2015;1319:155–175. DOI: 10.1007/978-1-4939-2748-7_8

[24] Cherf G.M., Cochran J.R. Applications of yeast surface display for protein engineering // Methods Mol Biol. 2015. Vol. 1319. P. 155–175. DOI: 10.1007/978-1-4939-2748-7_8

[25] Pack SP, Park K, Yoo YJ. Enhancement of β-glucosidase stability and cellobiose-usage using surface-engineered recombinant Saccharomyces cerevisiae in ethanol production. Biotechnology Letters. 2002;24:1919–1925. DOI: 10.1023/A:1020908426815

[26] Pack S.P., Park K., Yoo Y.J. Enhancement of β-glucosidase stability and cellobiose-usage using surface-engineered recombinant Saccharomyces cerevisiae in ethanol production // Biotechnology Letters. 2002. Vol. 24. P. 1919–1925. DOI: 10.1023/A:1020908426815

[27] Andreu C, Del Olmo ML. Yeast arming systems: pros and cons of different protein anchors and other elements required for display. Appl Microbiol Biotechnol. 2018;102(6):2543–2561. DOI: 10.1007/s00253-018-8827-6

[28] Andreu C., Del Olmo M.L. Yeast arming systems: pros and cons of different protein anchors and other elements required for display // Appl Microbiol Biotechnol. 2018. Vol. 102, No. 6. P. 2543–2561. DOI: 10.1007/s00253-018-8827-6

[29] Mattanovich D, Callewaert N, Rouzé P, et al. Open access to sequence: browsing the Pichia pastoris genome. Microb Cell Fact. 2009;8:53. DOI: 10.1186/1475-2859-8-53

[30] Mattanovich D., Callewaert N., Rouzé P., et al. Open access to sequence: browsing the Pichia pastoris genome // Microb Cell Fact. 2009. Vol. 8. ID53. DOI: 10.1186/1475-2859-8-53

[31] Cereghino JL, Cregg JM. Heterologous protein expression in the methylotrophic yeast Pichia pastoris. FEMS Microbiol Rev. 2000;24(1):45–66. DOI: 10.1111/j.1574-6976.2000.tb00532.x

[32] Cereghino J.L., Cregg J.M. Heterologous protein expression in the methylotrophic yeast Pichia pastoris // FEMS Microbiol Rev. 2000. Vol. 24, No. 1. P. 45–66. DOI: 10.1111/j.1574-6976.2000.tb00532.x

[33] Padkina MV, Sambuk EV. Prospects for the application of yeast display in biotechnology and cell biology (review). Applied Biochemistry and Microbiology. 2018;54(4):337–351. (In Russ.) DOI: 10.7868/S0555109918040013

[34] Падкина М.В., Самбук Е.В. Перспективы использования дрожжевого дисплея в биотехнологии и клеточной биологии (обзор) // Прикладная биохимия и микробиология. 2018. Т. 54, № 4. С. 337–351. DOI: 10.7868/S0555109918040013

[35] Ahmad M, Hirz M, Pichler H, Schwab H. Protein expression in Pichia pastoris: recent achievements and perspectives for heterologous protein production. Appl Microbiol Biotechnol. 2014;98(12): 5301–5317. DOI: 10.1007/s00253-014-5732-5

[36] Ahmad M., Hirz M., Pichler H., Schwab H. Protein expression in Pichia pastoris: recent achievements and perspectives for heterologous protein production // Appl Microbiol Biotechnol. 2014. Vol. 98, No. 12. P. 5301–5317. DOI: 10.1007/s00253-014-5732-5

[37] Prabhu AA, Boro B, Bharali B, et al. Gene and process level modulation to overcome the bottlenecks of recombinant proteins expression in Pichia pastoris. Curr Pharm Biotechnol. 2017;18(15): 1200–1223. DOI: 10.2174/1389201019666180329112827

[38] Prabhu A.A., Boro B., Bharali B., et al. Gene and process level modulation to overcome the bottlenecks of recombinant proteins expression in Pichia pastoris // Curr Pharm Biotechnol. 2017. Vol. 18, No. 15. P. 1200–1223. DOI: 10.2174/1389201019666180329112827

[39] Cereghino GPL, Cereghino JL, Ilgen C, Cregg JM. Production of recombinant proteins in fermenter cultures of the yeast Pichia pastoris. Curr Opin Biotechnol. 2002;13(4):329–332. DOI: 10.1016/S0958-1669(02)00330-0

[40] Cereghino G.P.L., Cereghino J.L., Ilgen C., Cregg J.M. Production of recombinant proteins in fermenter cultures of the yeast Pichia pastoris // Curr Opin Biotechnol. 2002. Vol. 13, No. 4. P. 329–332. DOI: 10.1016/S0958-1669(02)00330-0

[41] Gaboardi GC, Alves D, de los Santos DG, et al. Influence of Pichia pastoris X-33 produced in industrial residues on productive performance, egg quality, immunity, and intestinal morphometry in quails. Sci Rep. 2019;9(1):15372. DOI: 10.1038/s41598-019-51908-0

[42] Gaboardi G.C., Alves D., de los Santos D.G., et al. Influence of Pichia pastoris X-33 produced in industrial residues on productive performance, egg quality, immunity, and intestinal morphometry in quails // Sci Rep. 2019. Vol. 9, No. 1. ID 15372. DOI: 10.1038/s41598-019-51908-0

[43] Mergler M, Wolf K, Zimmermann M. Development of a bisphenol A-adsorbing yeast by surface display of the Kluyveromyces yellow enzyme on Pichia pastoris. Appl Microbiol Biotechnol. 2004;63(4):418–421. DOI: 10.1007/s00253-003-1361-0

[44] Mergler M., Wolf K., Zimmermann M. Development of a bisphenol A-adsorbing yeast by surface display of the Kluyveromyces yellow enzyme on Pichia pastoris // Appl Microbiol Biotechnol. 2004. Vol. 63, No. 4. P. 418–421. DOI: 10.1007/s00253-003-1361-0

[45] Wang Q, Li L, Chen M, et al. Construction of a novel system for cell surface display of heterologous proteins on Pichia pastoris. Biotechnol Lett. 2007;29(10):1561–1566. DOI: 10.1007/s10529-007-9430-6

[46] Wang Q., Li L., Chen M., et al. Construction of a novel system for cell surface display of heterologous proteins on Pichia pastoris // Biotechnol Lett. 2007. Vol. 29, No. 10. P. 1561–1566. DOI: 10.1007/s10529-007-9430-6

[47] Zhang L, Liang S, Zhou X, et al. Screening for glycosylphosphatidylinositol-modified cell wall proteins in Pichia pastoris and their recombinant expression on the cell surface. Appl Environ Microbiol. 2013;79(18):5519–5526. DOI: 10.1128/AEM.00824-13

[48] Zhang L., Liang S., Zhou X., et al. Screening for glycosylphosphatidylinositol-modified cell wall proteins in Pichia pastoris and their recombinant expression on the cell surface // Appl Environ Microbiol. 2013. Vol. 79, No. 18. P. 5519–5526. DOI: 10.1128/AEM.00824-13

[49] Yang J, Huang K, Xu X, et al. Cell surface display of Thermomyces lanuginosus lipase in Pichia pastoris. Front Bioeng Biotechnol. 2020;8:544058. DOI: 10.3389/fbioe.2020.544058

[50] Yang J., Huang K., Xu X., et al. Cell Surface Display of Thermomyces lanuginosus Lipase in Pichia pastoris // Front Bioeng Biotechnol. 2020. Vol. 8. ID544058. DOI: 10.3389/fbioe.2020.544058

[51] Matsuda T, Cepko CL. Electroporation and RNA interference in the rodent retina in vivo and in vitro. PNAS USA. 2004;101(1):16–22. DOI: 10.1073/pnas.2235688100

[52] Matsuda T., Cepko C.L. Electroporation and RNA interference in the rodent retina in vivo and in vitro // PNAS USA. 2004. Vol. 101, No. 1. P. 16–22. DOI: 10.1073/pnas.2235688100

[53] Wu S, Letchworth GJ. High efficiency transformation by electroporation of Pichia pastoris pretreated with lithium acetate and dithiothreitol. Biotechniques. 2004;36(1):152–154. DOI: 10.2144/04361DD02

[54] Wu S., Letchworth G.J. High efficiency transformation by electroporation of Pichia pastoris pretreated with lithium acetate and dithiothreitol // Biotechniques. 2004. Vol. 36, No. 1. P. 152–154. DOI: 10.2144/04361DD02

[55] Maniatis T, Frich E, Sembruk Dzh. Metody geneticheskoj inzhenerii. Molekulyarnoe klonirovanie. Moscow: Mir, 1984. (In Russ.)

[56] Маниатис Т., Фрич Э., Сэмбрук Дж. Методы генетической инженерии. Молекулярное клонирование. Москва: Мир, 1984.

[57] graphpad.com [Internet]. GraphPad Prism software [cited 2022 Oct 10]. Available at: https://www.graphpad.com/

[58] graphpad.com [Электронный ресурс]. GraphPad Prism software [дата обращения: 2022 Oct 10]. Доступ по ссылке: https://www.graphpad.com/

[59] Lipke PN, Kurjan J. Sexual agglutination in budding yeasts: structure, function, and regulation of adhesion glycoproteins. Microbiol Rev. 1992;56(1):180–194. DOI: 10.1128/mr.56.1.180-194.1992

[60] Lipke P.N., Kurjan J. Sexual agglutination in budding yeasts: structure, function, and regulation of adhesion glycoproteins // Microbiol Rev. 1992. Vol. 56, No. 1. P. 180–194. DOI: 10.1128/mr.56.1.180-194.1992

Funding

Санкт-Петербургский государственный университетSaint-Petersburg State University(75428571)

RIGHTS & PERMISSIONS

Tsygankov M.A., Rumyantsev A.M., Makeeva A.S., Padkina M.V.