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Information × Registration Number 0218U005114, 0115U001360 , R & D reports Title The mechanisms of the degradation of peroxisomal and cytosolic proteins in methylotrophic yeast and creation of the overprodusers of proteins with commercial value from mutants with damage of these mechanisms popup.stage_title Head Sibirny Andriy Andriyovych, Registration Date 01-03-2018 Organization Institute of cell biology, National Academy of Sciences of Ukraine popup.description2 The maximum reduction in the level of degradation of the target recombinant protein in the cytosol is a guarantee of the creation of superproducents of the proteins of industrial significance. The lack of convenient methods for analyzing P. pastoris mutants with truncated cytosolic proteins degradation directly in yeast colonies complicates not only the isolation of mutants, but also the identification of the relevant genes. However, such mutants can be analyzed on the model of the heterologous enzyme b-galactosidase, for which there are convenient methods of analysis. To this purpose, vector for expression of b-galactosidase (LAC4) from Kluveromyces lactis under the control of the methanol-regulated promoter pFLD1 was constructed. This vector was used to obtain P. pastoris strains with the expression of the heterologous protein b-galactosidase on the basis of the wild type GS200 strain, as well as the strain SMD1163 with damaged autophagy. Thus, P. pastoris strains with the expression of the b-galactosidase heterologous protein under the control of pFLD were obtained. These strains were verified by PCR analysis and qualitative determination of b-galactosidase was carried out under the conditions of induction by methanol of the promoter of FLD1 gene. When cultivating this strain in an environment with methanol and an analogue of lactose X-gal, or with a surface filler containing the ONPG, reaction mixture forms insoluble light blue or yellow dyes, respectively, indicating the presence of the active protein b-galactosidase. To study the b-galactosidase degradation, cells grown on methanol were transferred to the medium with glucose. It has been found that b-galactosidase is partially degraded by autophagy. In addition, vectors for expression of fructose-1,6-bisphosphatase from E. coli and P. pastoris under the control of the regulated promoter of the FBP1 gene or the constitutive promoter of the GAP1 gene have been constructed.5481 Product Description popup.authors Булботка Ніна Володимирівна Дмитрук Олена Валеріївна Левків Катерина Олександрівна Сибірний Андрій Андрійович popup.nrat_date 2020-04-02 Close