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Methylotrophic yeast Komagataella phaffii as Neoleukin producer
- Authors: Makeeva A.S.1, Shubert M.A.1, Al Shanaa O.1, Rumyantsev A.M.1
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Affiliations:
- Saint Petersburg State University
- Issue: Vol 20 (2022): Supplement
- Pages: 44-45
- Section: Genetically modified organism. The Нistory, Achivements, Social and Environmental Riscs
- URL: https://journals.eco-vector.com/ecolgenet/article/view/112318
- DOI: https://doi.org/10.17816/ecogen112318
- ID: 112318
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Abstract
Methylotrophic yeast Komagataella рhaffii (also known as Pichia pastoris) is widely applied in biotechnology for recombinant protein production. K. рhaffii particularly proved to be a successful host system for the synthesis of immunomodulators such as interferons [1].
In this study, we engineered K. рhaffii strains capable of producing the immunomodulatory protein Neoleukin (Neo-2/15). Neo-2/15 is an interleukin-2 mimetic, designed by in silico methods [2]. In preclinical studies on murine cancer models, Neo-2/15 showed superior therapeutic effect to inerleukin-2 with reduced toxicity.
In this work, we show that K. рhaffii can successfully synthesize and secrete Neo-2/15. We have obtained a number of K. phaffii strains, including MutS and Mut+, with different Neo-2/15 expression cassettes integrated into the genome, carrying up to five copies of Neo-2/15 gene. In fact, the higher number of Neo-2/15 gene copies in K. рhaffii genome allowed a higher protein yield.
In this study, we further developed a split marker approach [3] for yeast transformation using two DNA fragments, comprising of the expression cassette and the overlapping fragments of the marker gene. This allowed us to generate MutS strains with two copies using pPICZαB vector, which is not originally intended for MutS strain generation.
As a result, we demonstrated that K. phaffii is a perspective producer of Neo-2/15, providing wide opportunities to increase the production of this therapeutic protein.
Full Text
Methylotrophic yeast Komagataella рhaffii (also known as Pichia pastoris) is widely applied in biotechnology for recombinant protein production. K. рhaffii particularly proved to be a successful host system for the synthesis of immunomodulators such as interferons [1].
In this study, we engineered K. рhaffii strains capable of producing the immunomodulatory protein Neoleukin (Neo-2/15). Neo-2/15 is an interleukin-2 mimetic, designed by in silico methods [2]. In preclinical studies on murine cancer models, Neo-2/15 showed superior therapeutic effect to inerleukin-2 with reduced toxicity.
In this work, we show that K. рhaffii can successfully synthesize and secrete Neo-2/15. We have obtained a number of K. phaffii strains, including MutS and Mut+, with different Neo-2/15 expression cassettes integrated into the genome, carrying up to five copies of Neo-2/15 gene. In fact, the higher number of Neo-2/15 gene copies in K. рhaffii genome allowed a higher protein yield.
In this study, we further developed a split marker approach [3] for yeast transformation using two DNA fragments, comprising of the expression cassette and the overlapping fragments of the marker gene. This allowed us to generate MutS strains with two copies using pPICZαB vector, which is not originally intended for MutS strain generation.
As a result, we demonstrated that K. phaffii is a perspective producer of Neo-2/15, providing wide opportunities to increase the production of this therapeutic protein.
About the authors
Anastasia S. Makeeva
Saint Petersburg State University
Author for correspondence.
Email: anastasimakeeva@mail.ru
SPIN-code: 1412-8449
Research Engineer, Department of Microbiology
Russian Federation, Saint PetersburgMaria A. Shubert
Saint Petersburg State University
Email: marishubert01@gmail.com
Research Assistant, Department of Genetics and Biotechnology
Russian Federation, Saint PetersburgOusama Al Shanaa
Saint Petersburg State University
Email: st072427@student.spbu.ru
SPIN-code: 7453-9258
Junior Researcher, Department of Genetics and Biotechnology
Russian Federation, Saint PetersburgAndrei M. Rumyantsev
Saint Petersburg State University
Email: rumyantsev-am@mail.ru
SPIN-code: 9335-1184
PhD, Senior Researcher, Department of Genetics and Biotechnology
Russian Federation, Saint PetersburgReferences
- Tsygankov MA, Zobnina AE, Padkina MV. Synthesis of recombinant gamma interferons resistant to proteolysis in the yeast Pichia pastoris. Appl Biochem Microbiol. 2014;50(4):429–436. (In Russ.) doi: 10.7868/s055510991404028x
- Silva D-A, Yu S, Ulge UY, et al. De novo design of potent and selective mimics of IL-2 and IL-15. Nature. 2019;565(7738):186–191. doi: 10.1038/s41586-018-0830-7
- Heiss S, Maurer M, Hahn R, et al. Identification and deletion of the major secreted protein of Pichia pastoris. Appl Microbiol Biotechnol. 2013;97(3):1241–1249. doi: 10.1007/s00253-012-4260-4
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