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...

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Autores principales:	Lukas Verhülsdonk, Hans Georg Mannherz, Markus Napirei
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2021
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/40efb057740b4a66a1b30216ec15faed
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spelling	oai:doaj.org-article:40efb057740b4a66a1b30216ec15faed2021-12-02T20:08:51ZComparison of the secretory murine DNase1 family members expressed in Pichia pastoris.1932-620310.1371/journal.pone.0253476https://doaj.org/article/40efb057740b4a66a1b30216ec15faed2021-01-01T00:00:00Zhttps://doi.org/10.1371/journal.pone.0253476https://doaj.org/toc/1932-6203Soluble 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.Lukas VerhülsdonkHans Georg MannherzMarkus NapireiPublic Library of Science (PLoS)articleMedicineRScienceQENPLoS ONE, Vol 16, Iss 7, p e0253476 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Lukas Verhülsdonk Hans Georg Mannherz Markus Napirei Comparison of the secretory murine DNase1 family members expressed in Pichia pastoris.
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	article
author	Lukas Verhülsdonk Hans Georg Mannherz Markus Napirei
author_facet	Lukas Verhülsdonk Hans Georg Mannherz Markus Napirei
author_sort	Lukas Verhülsdonk
title	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_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_sort	comparison of the secretory murine dnase1 family members expressed in pichia pastoris.
publisher	Public Library of Science (PLoS)
publishDate	2021
url	https://doaj.org/article/40efb057740b4a66a1b30216ec15faed
work_keys_str_mv	AT lukasverhulsdonk comparisonofthesecretorymurinednase1familymembersexpressedinpichiapastoris AT hansgeorgmannherz comparisonofthesecretorymurinednase1familymembersexpressedinpichiapastoris AT markusnapirei comparisonofthesecretorymurinednase1familymembersexpressedinpichiapastoris
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Comparison of the secretory murine DNase1 family members expressed in Pichia pastoris.

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