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

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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.

About the authors

Miklhail A. Tsygankov

Saint Petersburg State University

Author for correspondence.
Email: mial.tsygankov@yandex.ru
ORCID iD: 0000-0002-2513-6655
SPIN-code: 1098-0995
Scopus Author ID: 56252740000

Engineer-Researcher, Faculty of Biology, Department of Genetics and Biotechnology, Laboratory of biochemical genetics

Russian Federation, Saint Petersburg

Andrey M. Rumyantsev

Saint Petersburg State University

Email: rumyantsev-am@mail.ru
ORCID iD: 0000-0002-1744-3890
SPIN-code: 9335-1184
Scopus Author ID: 55370658800

Cand. Sci. (Med.), Senior Research Associate, Faculty of Biology, Department of Genetics and Biotechnology, Laboratory of biochemical genetics

Russian Federation, Saint Petersburg

Anastasiya S. Makeeva

Saint Petersburg State University

Email: anastasimakeeva@mail.ru
SPIN-code: 1412-8449

Engineer-Researcher, Postgraduate Student, Faculty of Biology, Department of Genetics and Biotechnology, Laboratory of biochemical genetics

Russian Federation, Saint Petersburg

Marina V. Padkina

Saint Petersburg State University

Email: mpadkina@mail.ru
ORCID iD: 0000-0002-4051-4837
SPIN-code: 7709-0449
Scopus Author ID: 6602596755

Dr. Sci. (Biol.), Professor, Faculty of Biology, Department of Genetics and Biotechnology, Laboratory of biochemical genetics

Russian Federation, Saint Petersburg

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Supplementary files

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2. Fig. 1. Schematic representation of the plasmids used in the work. a — plasmid with the eGFP reporter gene; b — obtained plasmids with the eGFP gene and cell wall protein genes. 5'AOX1 — AOX1 gene promoter; α-factor — yeast alpha factor; PTEF1 — yeast promoter of the TEF1 gene; PEM7 — bacterial promoter; CYC1 TT — terminator region of the yeast CYC1 gene; Zeocin — zeocin resistance gene; pUC ori — origin replications; AOX1 TT — transcription termination region of the AOX1 gene; eGFP — reporter protein gene; ScAGα1l (long), ScAGα1, KpCW51, KpCW61 — cell wall proteins; XhoI, XbaI, AgeI — restriction enzyme recognition sites

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3. Fig. 2. Electrophoregram of PCR products with primers to the genes ScAGa1long, ScAGa1, KpCW51, KpCW61. M — marker NL001 (Eurogene, Russia); 1 — ScAGa1long (expected fragment — 1421 bp), 2 — ScAGa1 (expected fragment — 985 bp), 3 — KpCW51 (expected fragment — 612 bp), 4 — KpCW61 (expected fragment — 171 bp)

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4. Fig. 3. Electrophoregram of PCR products on a matrix of chromosomal DNA of yeast transformants with the ScAGα1long, ScAGα1, KpCW51, KpCW61 genes in the same reading frame with the eGFP reporter protein gene using reverse primers to the corresponding cell wall protein genes and a forward primer to the eGFP protein gene (1–3) or primer to the fragment of the AOX1 gene (4). M — NL001 marker (Evrogen, Russia); K+ — positive controls (plasmids used to transform the original yeast strain); K– — negative controls (PCR reaction mixtures not containing DNA templates); 1 — ScAGα1long (expected fragment – 2158 bp); 2 — ScAGα1 (expected fragment — 1717 bp); 3 — KpCW51 (expected fragment — 1344 bp), 4 — KpCW61 (expected fragment — 1225 bp)

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5. Fig. 4. Confocal microscopy of obtained strains with eGFP reporter protein. Cells of the original strain K. phaffii X-33 were used as a control variant (K–)

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6. Fig. 5. Cells fluorescence with the eGFP protein (a) and dye-labeled Cy3 antibodies to the eGFP protein (b). Data are presented with standard error of the mean. K– — original strain X-33

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Copyright (c) 2022 Tsygankov M.A., Rumyantsev A.M., Makeeva A.S., Padkina M.V.

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


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