Identification of a Caldariomyces fumago Mutant Secreting an Inactive Form of Chloroperoxidase Lacking the Heme Group and N-Glycans

Identification of a Caldariomyces fumago Mutant Secreting an Inactive Form of Chloroperoxidase Lacking the Heme Group and N-Glycans

By mutant colony screening of Caldariomyces fumago a mutant was isolated which was slightly greenish on fructose minimal medium and grew slower in comparison to the wild type. The supernatant samples lacked the Soret band typical for the heme group of the CPO and nearly no CPO activity was detected....

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Main Authors: Hüttmann, Sonja, Buchhaupt, Markus, Schrader, Jens
Format: Online
Language:English
Published: Public Library of Science 2013
Online Access:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3699481/
id pubmed-3699481
recordtype oai_dc
spelling pubmed-36994812013-07-10 Identification of a Caldariomyces fumago Mutant Secreting an Inactive Form of Chloroperoxidase Lacking the Heme Group and N-Glycans Hüttmann, Sonja Buchhaupt, Markus Schrader, Jens Research Article By mutant colony screening of Caldariomyces fumago a mutant was isolated which was slightly greenish on fructose minimal medium and grew slower in comparison to the wild type. The supernatant samples lacked the Soret band typical for the heme group of the CPO and nearly no CPO activity was detected. SDS-PAGE analysis of mutant culture supernatant samples showed production of a 38–40 kDa protein while wild type samples contain the 42 kDa CPO protein. Protein identification using nanoLC-ESI-MS/MS was performed and based on three peptides the protein in the mutant culture was identified as CPO. No differences in the CPO gene sequences of wild type and mutant were found indicating a post-translational defect in protein maturation. Deglycosylation experiments using CPO from wild type and mutant were carried out. After removing N-linked oligosaccharides from wild type CPO a protein band at 38–40 kDa was detected. Our results reveal that the mutant protein lacks the heme group as well as the N-glycans. Public Library of Science 2013-07-02 /pmc/articles/PMC3699481/ /pubmed/23844113 http://dx.doi.org/10.1371/journal.pone.0067857 Text en © 2013 Schrader et al http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.
repository_type Open Access Journal
institution_category Foreign Institution
institution US National Center for Biotechnology Information
building NCBI PubMed
collection Online Access
language English
format Online
author Hüttmann, Sonja
Buchhaupt, Markus
Schrader, Jens
spellingShingle Hüttmann, Sonja
Buchhaupt, Markus
Schrader, Jens
Identification of a Caldariomyces fumago Mutant Secreting an Inactive Form of Chloroperoxidase Lacking the Heme Group and N-Glycans
author_facet Hüttmann, Sonja
Buchhaupt, Markus
Schrader, Jens
author_sort Hüttmann, Sonja
title Identification of a Caldariomyces fumago Mutant Secreting an Inactive Form of Chloroperoxidase Lacking the Heme Group and N-Glycans
title_short Identification of a Caldariomyces fumago Mutant Secreting an Inactive Form of Chloroperoxidase Lacking the Heme Group and N-Glycans
title_full Identification of a Caldariomyces fumago Mutant Secreting an Inactive Form of Chloroperoxidase Lacking the Heme Group and N-Glycans
title_fullStr Identification of a Caldariomyces fumago Mutant Secreting an Inactive Form of Chloroperoxidase Lacking the Heme Group and N-Glycans
title_full_unstemmed Identification of a Caldariomyces fumago Mutant Secreting an Inactive Form of Chloroperoxidase Lacking the Heme Group and N-Glycans
title_sort identification of a caldariomyces fumago mutant secreting an inactive form of chloroperoxidase lacking the heme group and n-glycans
description By mutant colony screening of Caldariomyces fumago a mutant was isolated which was slightly greenish on fructose minimal medium and grew slower in comparison to the wild type. The supernatant samples lacked the Soret band typical for the heme group of the CPO and nearly no CPO activity was detected. SDS-PAGE analysis of mutant culture supernatant samples showed production of a 38–40 kDa protein while wild type samples contain the 42 kDa CPO protein. Protein identification using nanoLC-ESI-MS/MS was performed and based on three peptides the protein in the mutant culture was identified as CPO. No differences in the CPO gene sequences of wild type and mutant were found indicating a post-translational defect in protein maturation. Deglycosylation experiments using CPO from wild type and mutant were carried out. After removing N-linked oligosaccharides from wild type CPO a protein band at 38–40 kDa was detected. Our results reveal that the mutant protein lacks the heme group as well as the N-glycans.
publisher Public Library of Science
publishDate 2013
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3699481/
_version_ 1611991206200344576

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