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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Bibliographic Details
Published in:PloS one 2017-02, Vol.12 (2), p.e0172712-e0172712
Main Authors: Jin, Hyeon-Su, Park, Seon Yeong, Kim, Kyungmin, Lee, Yong-Jik, Nam, Gae-Won, Kang, Nam Joo, Lee, Dong-Woo
Format: Article
Language:English
Subjects:
Affinity chromatography
Animals
Assaying
Bacillus subtilis
Biology and Life Sciences
Casein
Chickens
Chromatography
Chromosomes
Dialysis
E coli
Endopeptidase K
Enzymes
Escherichia coli
Feathers
Feathers - metabolism
Gallus gallus
Genomes
Hair
High-throughput screening
High-throughput screening (Biochemical assaying)
Hydrolysates
Keratin
Keratinase
Keratinocytes
Keratins
Keratins - metabolism
Papain
Peptide Hydrolases - analysis
Pets
Polymers
Poultry
Proteinase
Proteins
Proteolysis
Red junglefowl
Research and Analysis Methods
Substrate specificity
Substrates
Sulfur content
Tandem Mass Spectrometry
Trypsin
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Summary: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 Ni2+ 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.
ISSN:1932-6203
1932-6203
DOI:10.1371/journal.pone.0172712