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Disruption of YPS1 and PEP4 genes reduces proteolytic degradation of secreted HSA/PTH in Pichia pastoris GS115
Human serum albumin (HSA) and human parathyroid hormone (1-34) [PTH (1-34)] fusion protein [HSA/PTH (1-34)] is a promising long-acting form of PTH (1-34) for osteoporosis treatment. Secretory expression of intact HSA/PTH (1-34) in Pichia pastoris GS115 was accompanied by two degradation fragments, w...
Autores principales: | , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Springer-Verlag
2013
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3656227/ https://www.ncbi.nlm.nih.gov/pubmed/23529666 http://dx.doi.org/10.1007/s10295-013-1264-8 |
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author | Wu, Min Shen, Qi Yang, Yong Zhang, Sheng Qu, Wen Chen, Jing Sun, Hongying Chen, Shuqing |
author_facet | Wu, Min Shen, Qi Yang, Yong Zhang, Sheng Qu, Wen Chen, Jing Sun, Hongying Chen, Shuqing |
author_sort | Wu, Min |
collection | PubMed |
description | Human serum albumin (HSA) and human parathyroid hormone (1-34) [PTH (1-34)] fusion protein [HSA/PTH (1-34)] is a promising long-acting form of PTH (1-34) for osteoporosis treatment. Secretory expression of intact HSA/PTH (1-34) in Pichia pastoris GS115 was accompanied by two degradation fragments, with molecular weights around 66 kDa, in addition to the well-known ~45 kDa HSA-truncated fragment, resulting in a low yield of intact protein. In this study, two internal cleavage sites were identified in the PTH (1-34) portion of the fusion protein by Western Blot analysis. To minimize proteolytic cleavages, several protease genes including PEP4 (encoding proteinase A), PRB1 (proteinase B) and seven YPSs genes (yapsin family members) were knocked out respectively by disruption of the individual genes and the selective combinations. Reduced degradation was observed by single disruption of either PEP4 gene or YPS1 gene, and the lowest level of degradation was observed in a pep4△yps1△ double disruptant. After 72 h of induction, more than 80 % of the HSA/PTH (1-34) secreted by the pep4△yps1△ double disruptant remained intact, in comparison to only 30 % with the wild-type strain. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s10295-013-1264-8) contains supplementary material, which is available to authorized users. |
format | Online Article Text |
id | pubmed-3656227 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2013 |
publisher | Springer-Verlag |
record_format | MEDLINE/PubMed |
spelling | pubmed-36562272013-05-17 Disruption of YPS1 and PEP4 genes reduces proteolytic degradation of secreted HSA/PTH in Pichia pastoris GS115 Wu, Min Shen, Qi Yang, Yong Zhang, Sheng Qu, Wen Chen, Jing Sun, Hongying Chen, Shuqing J Ind Microbiol Biotechnol Biotechnology Methods Human serum albumin (HSA) and human parathyroid hormone (1-34) [PTH (1-34)] fusion protein [HSA/PTH (1-34)] is a promising long-acting form of PTH (1-34) for osteoporosis treatment. Secretory expression of intact HSA/PTH (1-34) in Pichia pastoris GS115 was accompanied by two degradation fragments, with molecular weights around 66 kDa, in addition to the well-known ~45 kDa HSA-truncated fragment, resulting in a low yield of intact protein. In this study, two internal cleavage sites were identified in the PTH (1-34) portion of the fusion protein by Western Blot analysis. To minimize proteolytic cleavages, several protease genes including PEP4 (encoding proteinase A), PRB1 (proteinase B) and seven YPSs genes (yapsin family members) were knocked out respectively by disruption of the individual genes and the selective combinations. Reduced degradation was observed by single disruption of either PEP4 gene or YPS1 gene, and the lowest level of degradation was observed in a pep4△yps1△ double disruptant. After 72 h of induction, more than 80 % of the HSA/PTH (1-34) secreted by the pep4△yps1△ double disruptant remained intact, in comparison to only 30 % with the wild-type strain. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s10295-013-1264-8) contains supplementary material, which is available to authorized users. Springer-Verlag 2013-03-26 2013 /pmc/articles/PMC3656227/ /pubmed/23529666 http://dx.doi.org/10.1007/s10295-013-1264-8 Text en © The Author(s) 2013 https://creativecommons.org/licenses/by/2.0/ Open AccessThis article is distributed under the terms of the Creative Commons Attribution License which permits any use, distribution, and reproduction in any medium, provided the original author(s) and the source are credited. |
spellingShingle | Biotechnology Methods Wu, Min Shen, Qi Yang, Yong Zhang, Sheng Qu, Wen Chen, Jing Sun, Hongying Chen, Shuqing Disruption of YPS1 and PEP4 genes reduces proteolytic degradation of secreted HSA/PTH in Pichia pastoris GS115 |
title | Disruption of YPS1 and PEP4 genes reduces proteolytic degradation of secreted HSA/PTH in Pichia pastoris GS115 |
title_full | Disruption of YPS1 and PEP4 genes reduces proteolytic degradation of secreted HSA/PTH in Pichia pastoris GS115 |
title_fullStr | Disruption of YPS1 and PEP4 genes reduces proteolytic degradation of secreted HSA/PTH in Pichia pastoris GS115 |
title_full_unstemmed | Disruption of YPS1 and PEP4 genes reduces proteolytic degradation of secreted HSA/PTH in Pichia pastoris GS115 |
title_short | Disruption of YPS1 and PEP4 genes reduces proteolytic degradation of secreted HSA/PTH in Pichia pastoris GS115 |
title_sort | disruption of yps1 and pep4 genes reduces proteolytic degradation of secreted hsa/pth in pichia pastoris gs115 |
topic | Biotechnology Methods |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3656227/ https://www.ncbi.nlm.nih.gov/pubmed/23529666 http://dx.doi.org/10.1007/s10295-013-1264-8 |
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