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Registration Number

0224U032662, (0124U004350) , R & D reports

Title

Construction of yeast producers of granulocyte-macrophage colony-stimulating factor (GM-CSF) activating wound healing

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Дизайн нуклеотидних послідовностей, що кодують ГМ-КСФ, та конструювання відповідних векторів для експресії як секреторної форми ГМ-КСФ, так і форм для іммобілізації ГМ-КСФ на поверхні дріжджових клітин.

Head

Dmytruk Kostyantyn V., Доктор біологічних наук

Registration Date

29-11-2024

Organization

Institute of Cell Biology of the National Academy of Sciences of Ukraine

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The main goal of the project is to construct GM-CSF producers based on methylotrophic yeast with "humanized" protein glycosylation. Cultivation conditions of the obtained producers will be optimized to maximize GM-CSF production. Methods for GM-CSF purification will be developed. An additional objective is the immobilization of GM-CSF on the yeast cell surface. The activity of GM-CSF and yeast displaying this growth factor on surface will be evaluated by stimulating TF1 cell line proliferation.

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The primary objective of this project is to develop GM-CSF producers using the methylotrophic yeasts Komagataella phaffii and Ogataea polymorpha, engineered to have a “humanized” glycosylation pattern. To maximize GM-CSF production, cultivation conditions for these producers will be optimized, and effective methods for GM-CSF purification will be established. An additional aim is to immobilize GM-CSF on the surface of yeast cells. This approach, involving cell preparations with surface-anchored GM-CSF, has the potential to significantly reduce the production costs of drugs for burn treatment. As a result of the first stage of the project, integrative vectors have been constructed. These vectors contain sequences of a synthetic gene encoding human GM-CSF, optimized for yeast codon usage. They also include the α-secretion signal and anchor sequences from Saccharomyces cerevisiae. Two sets of vectors were designed, incorporating AOX gene homologous promoters to enable gene expression in K. phaffii and O. polymorpha. Each set includes plasmids designed to express either a secretory form of GM-CSF or a surface-anchored variant. The constructed vectors will be used to generate yeast transformants, followed by detailed analysis and optimization. This work represents a step forward in creating cost-effective treatments for burns and other medical conditions.

Product Description

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Dmytruk Olena V.
Sybirnyi Andrii A.

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2024-11-29