Creation of a recombinant Komagataella phaffii strain, a producer of proteinase K from Tritirachium album
The objects of the study were recombinant clones of Komagataella phaffii K51 carrying the heterologous proteinase K (PK-w) gene from Tritirachium album integrated into their genome as well as samples of recombinant proteinase K isolated from these clones. The aims of this work were i) to determine w...
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Siberian Branch of the Russian Academy of Sciences, Federal Research Center Institute of Cytology and Genetics, The Vavilov Society of Geneticists and Breeders
2022-01-01
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| Series: | Вавиловский журнал генетики и селекции |
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| Online Access: | https://vavilov.elpub.ru/jour/article/view/3207 |
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| author | A. B. Beklemishev M. B. Pykhtina Ya. M. Kulikov T. N. Goryachkovskaya D. V. Bochkov S. V. Sergeeva A. R. Vasileva V. P. Romanov D. S. Novikova S. E. Peltek |
| author_facet | A. B. Beklemishev M. B. Pykhtina Ya. M. Kulikov T. N. Goryachkovskaya D. V. Bochkov S. V. Sergeeva A. R. Vasileva V. P. Romanov D. S. Novikova S. E. Peltek |
| author_sort | A. B. Beklemishev |
| collection | DOAJ |
| description | The objects of the study were recombinant clones of Komagataella phaffii K51 carrying the heterologous proteinase K (PK-w) gene from Tritirachium album integrated into their genome as well as samples of recombinant proteinase K isolated from these clones. The aims of this work were i) to determine whether it is possible to create recombinant K. phaffii K51 clones overexpressing functionally active proteinase K from T. album and ii) to analyze the enzymatic activity of the resulting recombinant enzyme. The following methods were used: computational analysis of primary structure of the proteinase K gene, molecular biological methods (PCR, electrophoresis of DNA in an agarose gel, electrophoresis of proteins in an SDS polyacrylamide gel under denaturing conditions, spectrophotometry, and quantitative assays of protease activity), and genetic engineering techniques (cloning and selection of genes in bacterial cells Escherichia coli TOP10 and in the methylotrophic yeast K. phaffii K51). The gene encoding natural proteinase K (PK-w) was designed and optimized for expression in K. phaffii K51. The proteinase K gene was synthesized and cloned within the plasmid pPICZα-A vector in E. coli TOP10 cells. The proteinase K gene was inserted into pPICZα-A in such a way that – at a subsequent stage of transfection into yeast cells – it was efficiently expressed under the control of the promoter and terminator of the AOX1 gene, and the product of the exogenous gene contained the signal peptide of the Saccharomyces cerevisiae a-factor to ensure the protein’s secretion into the culture medium. The resultant recombinant plasmid (pPICZα-A/PK-w) was transfected into K. phaffii K51 cells. A recombinant K. phaffii K51 clone was obtained that carried the synthetic proteinase K gene and ensured its effective expression and secretion into the culture medium. An approximate productivity of the yeast recombinant clones for recombinant proteinase K was 25 μg/ mL after 4 days of cultivation. The resulting recombinant protease has a high specific proteolytic activity: ~5000 U/mg. |
| format | Article |
| id | doaj-art-256cd5d7107e4cd29a9e8ca6b6c31b15 |
| institution | Kabale University |
| issn | 2500-3259 |
| language | English |
| publishDate | 2022-01-01 |
| publisher | Siberian Branch of the Russian Academy of Sciences, Federal Research Center Institute of Cytology and Genetics, The Vavilov Society of Geneticists and Breeders |
| record_format | Article |
| series | Вавиловский журнал генетики и селекции |
| spelling | doaj-art-256cd5d7107e4cd29a9e8ca6b6c31b152025-02-01T09:58:10ZengSiberian Branch of the Russian Academy of Sciences, Federal Research Center Institute of Cytology and Genetics, The Vavilov Society of Geneticists and BreedersВавиловский журнал генетики и селекции2500-32592022-01-0125888288810.18699/VJ21.1021227Creation of a recombinant Komagataella phaffii strain, a producer of proteinase K from Tritirachium albumA. B. Beklemishev0M. B. Pykhtina1Ya. M. Kulikov2T. N. Goryachkovskaya3D. V. Bochkov4S. V. Sergeeva5A. R. Vasileva6V. P. Romanov7D. S. Novikova8S. E. Peltek9Federal Research Center of Fundamental and Translational Medicine; Institute of Cytology and Genetics of the Siberian Branch of the Russian Academy of SciencesFederal Research Center of Fundamental and Translational Medicine; Institute of Cytology and Genetics of the Siberian Branch of the Russian Academy of SciencesFederal Research Center of Fundamental and Translational Medicine; Institute of Cytology and Genetics of the Siberian Branch of the Russian Academy of SciencesInstitute of Cytology and Genetics of the Siberian Branch of the Russian Academy of SciencesInstitute of Cytology and Genetics of the Siberian Branch of the Russian Academy of SciencesInstitute of Cytology and Genetics of the Siberian Branch of the Russian Academy of SciencesInstitute of Cytology and Genetics of the Siberian Branch of the Russian Academy of SciencesInstitute of Cytology and Genetics of the Siberian Branch of the Russian Academy of SciencesEfirnoe Joint-Stock CompanyInstitute of Cytology and Genetics of the Siberian Branch of the Russian Academy of SciencesThe objects of the study were recombinant clones of Komagataella phaffii K51 carrying the heterologous proteinase K (PK-w) gene from Tritirachium album integrated into their genome as well as samples of recombinant proteinase K isolated from these clones. The aims of this work were i) to determine whether it is possible to create recombinant K. phaffii K51 clones overexpressing functionally active proteinase K from T. album and ii) to analyze the enzymatic activity of the resulting recombinant enzyme. The following methods were used: computational analysis of primary structure of the proteinase K gene, molecular biological methods (PCR, electrophoresis of DNA in an agarose gel, electrophoresis of proteins in an SDS polyacrylamide gel under denaturing conditions, spectrophotometry, and quantitative assays of protease activity), and genetic engineering techniques (cloning and selection of genes in bacterial cells Escherichia coli TOP10 and in the methylotrophic yeast K. phaffii K51). The gene encoding natural proteinase K (PK-w) was designed and optimized for expression in K. phaffii K51. The proteinase K gene was synthesized and cloned within the plasmid pPICZα-A vector in E. coli TOP10 cells. The proteinase K gene was inserted into pPICZα-A in such a way that – at a subsequent stage of transfection into yeast cells – it was efficiently expressed under the control of the promoter and terminator of the AOX1 gene, and the product of the exogenous gene contained the signal peptide of the Saccharomyces cerevisiae a-factor to ensure the protein’s secretion into the culture medium. The resultant recombinant plasmid (pPICZα-A/PK-w) was transfected into K. phaffii K51 cells. A recombinant K. phaffii K51 clone was obtained that carried the synthetic proteinase K gene and ensured its effective expression and secretion into the culture medium. An approximate productivity of the yeast recombinant clones for recombinant proteinase K was 25 μg/ mL after 4 days of cultivation. The resulting recombinant protease has a high specific proteolytic activity: ~5000 U/mg.https://vavilov.elpub.ru/jour/article/view/3207proteinase kgene cloningkomagataella phaffiigene expressionenzymatic activity |
| spellingShingle | A. B. Beklemishev M. B. Pykhtina Ya. M. Kulikov T. N. Goryachkovskaya D. V. Bochkov S. V. Sergeeva A. R. Vasileva V. P. Romanov D. S. Novikova S. E. Peltek Creation of a recombinant Komagataella phaffii strain, a producer of proteinase K from Tritirachium album Вавиловский журнал генетики и селекции proteinase k gene cloning komagataella phaffii gene expression enzymatic activity |
| title | Creation of a recombinant Komagataella phaffii strain, a producer of proteinase K from Tritirachium album |
| title_full | Creation of a recombinant Komagataella phaffii strain, a producer of proteinase K from Tritirachium album |
| title_fullStr | Creation of a recombinant Komagataella phaffii strain, a producer of proteinase K from Tritirachium album |
| title_full_unstemmed | Creation of a recombinant Komagataella phaffii strain, a producer of proteinase K from Tritirachium album |
| title_short | Creation of a recombinant Komagataella phaffii strain, a producer of proteinase K from Tritirachium album |
| title_sort | creation of a recombinant komagataella phaffii strain a producer of proteinase k from tritirachium album |
| topic | proteinase k gene cloning komagataella phaffii gene expression enzymatic activity |
| url | https://vavilov.elpub.ru/jour/article/view/3207 |
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