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Development of a keratinase activity assay using recombinant chicken feather keratin substrates

Poultry feathers consist mainly of the protein keratin, which is rich in β-pleated sheets and consequently resistant to proteolysis. Although many keratinases have been identified, the reasons for their substrate specificity towards β-keratin remain unclear due to difficulties in preparing a soluble...

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Autores principales: Jin, Hyeon-Su, Park, Seon Yeong, Kim, Kyungmin, Lee, Yong-Jik, Nam, Gae-Won, Kang, Nam Joo, Lee, Dong-Woo
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2017
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5322917/
https://www.ncbi.nlm.nih.gov/pubmed/28231319
http://dx.doi.org/10.1371/journal.pone.0172712
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author Jin, Hyeon-Su
Park, Seon Yeong
Kim, Kyungmin
Lee, Yong-Jik
Nam, Gae-Won
Kang, Nam Joo
Lee, Dong-Woo
author_facet Jin, Hyeon-Su
Park, Seon Yeong
Kim, Kyungmin
Lee, Yong-Jik
Nam, Gae-Won
Kang, Nam Joo
Lee, Dong-Woo
author_sort Jin, Hyeon-Su
collection PubMed
description Poultry feathers consist mainly of the protein keratin, which is rich in β-pleated sheets and consequently resistant to proteolysis. Although many keratinases have been identified, the reasons for their substrate specificity towards β-keratin remain unclear due to difficulties in preparing a soluble feather keratin substrate for use in activity assays. In the present study, we overexpressed Gallus gallus chromosomes 2 and 27 β-keratin-encoding genes in Escherichia coli, purified denatured recombinant proteins by Ni(2+) affinity chromatography, and refolded by stepwise dialysis to yield soluble keratins. To assess the keratinolytic activity, we compared the proteolytic activity of crude extracts from the feather- degrading bacterium Fervidobacterium islandicum AW-1 with proteinase K, trypsin, and papain using purified recombinant keratin and casein as substrates. All tested proteases showed strong proteolytic activities for casein, whereas only F. islandicum AW-1 crude extracts and proteinase K exhibited pronounced keratinolytic activity for the recombinant keratin. Moreover, LC-MS/MS analysis of keratin hydrolysates allowed us to predict the P1 sites of keratinolytic enzymes in the F. islandicum AW-1 extracts, thereby qualifying and quantifying the extent of keratinolysis. The soluble keratin-based assay has clear therapeutic and industrial potential for the development of a high-throughput screening system for proteases hydrolyzing disease-related protein aggregates, as well as mechanically resilient keratin-based polymers.
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spelling pubmed-53229172017-03-09 Development of a keratinase activity assay using recombinant chicken feather keratin substrates Jin, Hyeon-Su Park, Seon Yeong Kim, Kyungmin Lee, Yong-Jik Nam, Gae-Won Kang, Nam Joo Lee, Dong-Woo PLoS One Research Article Poultry feathers consist mainly of the protein keratin, which is rich in β-pleated sheets and consequently resistant to proteolysis. Although many keratinases have been identified, the reasons for their substrate specificity towards β-keratin remain unclear due to difficulties in preparing a soluble feather keratin substrate for use in activity assays. In the present study, we overexpressed Gallus gallus chromosomes 2 and 27 β-keratin-encoding genes in Escherichia coli, purified denatured recombinant proteins by Ni(2+) affinity chromatography, and refolded by stepwise dialysis to yield soluble keratins. To assess the keratinolytic activity, we compared the proteolytic activity of crude extracts from the feather- degrading bacterium Fervidobacterium islandicum AW-1 with proteinase K, trypsin, and papain using purified recombinant keratin and casein as substrates. All tested proteases showed strong proteolytic activities for casein, whereas only F. islandicum AW-1 crude extracts and proteinase K exhibited pronounced keratinolytic activity for the recombinant keratin. Moreover, LC-MS/MS analysis of keratin hydrolysates allowed us to predict the P1 sites of keratinolytic enzymes in the F. islandicum AW-1 extracts, thereby qualifying and quantifying the extent of keratinolysis. The soluble keratin-based assay has clear therapeutic and industrial potential for the development of a high-throughput screening system for proteases hydrolyzing disease-related protein aggregates, as well as mechanically resilient keratin-based polymers. Public Library of Science 2017-02-23 /pmc/articles/PMC5322917/ /pubmed/28231319 http://dx.doi.org/10.1371/journal.pone.0172712 Text en © 2017 Jin 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 (http://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
Jin, Hyeon-Su
Park, Seon Yeong
Kim, Kyungmin
Lee, Yong-Jik
Nam, Gae-Won
Kang, Nam Joo
Lee, Dong-Woo
Development of a keratinase activity assay using recombinant chicken feather keratin substrates
title Development of a keratinase activity assay using recombinant chicken feather keratin substrates
title_full Development of a keratinase activity assay using recombinant chicken feather keratin substrates
title_fullStr Development of a keratinase activity assay using recombinant chicken feather keratin substrates
title_full_unstemmed Development of a keratinase activity assay using recombinant chicken feather keratin substrates
title_short Development of a keratinase activity assay using recombinant chicken feather keratin substrates
title_sort development of a keratinase activity assay using recombinant chicken feather keratin substrates
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5322917/
https://www.ncbi.nlm.nih.gov/pubmed/28231319
http://dx.doi.org/10.1371/journal.pone.0172712
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