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Comparison of the secretory murine DNase1 family members expressed in Pichia pastoris
Soluble nucleases of the deoxyribonuclease 1 (DNase1) family facilitate DNA and chromatin disposal (chromatinolysis) during certain forms of cell differentiation and death and participate in the suppression of anti-nuclear autoimmunity as well as thrombotic microangiopathies caused by aggregated neu...
Autores principales: | , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Public Library of Science
2021
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8323900/ https://www.ncbi.nlm.nih.gov/pubmed/34329318 http://dx.doi.org/10.1371/journal.pone.0253476 |
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author | Verhülsdonk, Lukas Mannherz, Hans Georg Napirei, Markus |
author_facet | Verhülsdonk, Lukas Mannherz, Hans Georg Napirei, Markus |
author_sort | Verhülsdonk, Lukas |
collection | PubMed |
description | Soluble nucleases of the deoxyribonuclease 1 (DNase1) family facilitate DNA and chromatin disposal (chromatinolysis) during certain forms of cell differentiation and death and participate in the suppression of anti-nuclear autoimmunity as well as thrombotic microangiopathies caused by aggregated neutrophil extracellular traps. Since a systematic and direct comparison of the specific activities and properties of the secretory DNase1 family members is still missing, we expressed and purified recombinant murine DNase1 (rmDNase1), DNase1-like 2 (rmDNase1L2) and DNase1-like 3 (rmDNase1L3) using Pichia pastoris. Employing different strategies for optimizing culture and purification conditions, we achieved yields of pure protein between ~3 mg/l (rmDNase1L2 and rmDNase1L3) and ~9 mg/l (rmDNase1) expression medium. Furthermore, we established a procedure for post-expressional maturation of pre-mature DNase still bound to an unprocessed tri-N-glycosylated pro-peptide of the yeast α-mating factor. We analyzed glycosylation profiles and determined specific DNase activities by the hyperchromicity assay. Additionally, we evaluated substrate specificities under various conditions at equimolar DNase isoform concentrations by lambda DNA and chromatin digestion assays in the presence and absence of heparin and monomeric skeletal muscle α-actin. Our results suggest that due to its biochemical properties mDNase1L2 can be regarded as an evolutionary intermediate isoform of mDNase1 and mDNase1L3. Consequently, our data show that the secretory DNase1 family members complement each other to achieve optimal DNA degradation and chromatinolysis under a broad spectrum of biological conditions. |
format | Online Article Text |
id | pubmed-8323900 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2021 |
publisher | Public Library of Science |
record_format | MEDLINE/PubMed |
spelling | pubmed-83239002021-07-31 Comparison of the secretory murine DNase1 family members expressed in Pichia pastoris Verhülsdonk, Lukas Mannherz, Hans Georg Napirei, Markus PLoS One Research Article Soluble nucleases of the deoxyribonuclease 1 (DNase1) family facilitate DNA and chromatin disposal (chromatinolysis) during certain forms of cell differentiation and death and participate in the suppression of anti-nuclear autoimmunity as well as thrombotic microangiopathies caused by aggregated neutrophil extracellular traps. Since a systematic and direct comparison of the specific activities and properties of the secretory DNase1 family members is still missing, we expressed and purified recombinant murine DNase1 (rmDNase1), DNase1-like 2 (rmDNase1L2) and DNase1-like 3 (rmDNase1L3) using Pichia pastoris. Employing different strategies for optimizing culture and purification conditions, we achieved yields of pure protein between ~3 mg/l (rmDNase1L2 and rmDNase1L3) and ~9 mg/l (rmDNase1) expression medium. Furthermore, we established a procedure for post-expressional maturation of pre-mature DNase still bound to an unprocessed tri-N-glycosylated pro-peptide of the yeast α-mating factor. We analyzed glycosylation profiles and determined specific DNase activities by the hyperchromicity assay. Additionally, we evaluated substrate specificities under various conditions at equimolar DNase isoform concentrations by lambda DNA and chromatin digestion assays in the presence and absence of heparin and monomeric skeletal muscle α-actin. Our results suggest that due to its biochemical properties mDNase1L2 can be regarded as an evolutionary intermediate isoform of mDNase1 and mDNase1L3. Consequently, our data show that the secretory DNase1 family members complement each other to achieve optimal DNA degradation and chromatinolysis under a broad spectrum of biological conditions. Public Library of Science 2021-07-30 /pmc/articles/PMC8323900/ /pubmed/34329318 http://dx.doi.org/10.1371/journal.pone.0253476 Text en © 2021 Verhülsdonk et al https://creativecommons.org/licenses/by/4.0/This is an open access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. |
spellingShingle | Research Article Verhülsdonk, Lukas Mannherz, Hans Georg Napirei, Markus Comparison of the secretory murine DNase1 family members expressed in Pichia pastoris |
title | Comparison of the secretory murine DNase1 family members expressed in Pichia pastoris |
title_full | Comparison of the secretory murine DNase1 family members expressed in Pichia pastoris |
title_fullStr | Comparison of the secretory murine DNase1 family members expressed in Pichia pastoris |
title_full_unstemmed | Comparison of the secretory murine DNase1 family members expressed in Pichia pastoris |
title_short | Comparison of the secretory murine DNase1 family members expressed in Pichia pastoris |
title_sort | comparison of the secretory murine dnase1 family members expressed in pichia pastoris |
topic | Research Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8323900/ https://www.ncbi.nlm.nih.gov/pubmed/34329318 http://dx.doi.org/10.1371/journal.pone.0253476 |
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