Molecular cloning and characterization of novel Morus alba germin-like protein gene which encodes for a silkworm gut digestion-resistant antimicrobial protein.
<h4>Background</h4>Silkworm fecal matter is considered one of the richest sources of antimicrobial and antiviral protein (substances) and such economically feasible and eco-friendly proteins acting as secondary metabolites from the insect system can be explored for their practical utilit...
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doaj-03e2469817f4457888f1201b2dcc7cb72021-03-03T23:55:24ZengPublic Library of Science (PLoS)PLoS ONE1932-62032012-01-01712e5090010.1371/journal.pone.0050900Molecular cloning and characterization of novel Morus alba germin-like protein gene which encodes for a silkworm gut digestion-resistant antimicrobial protein.Bharat Bhusan PatnaikDong Hyun KimSeung Han OhYong-Su SongNguyen Dang Minh ChanhJong Sun KimWoo-jin JungAtul Kumar SahaBharat Bhushan BindrooYeon Soo Han<h4>Background</h4>Silkworm fecal matter is considered one of the richest sources of antimicrobial and antiviral protein (substances) and such economically feasible and eco-friendly proteins acting as secondary metabolites from the insect system can be explored for their practical utility in conferring broad spectrum disease resistance against pathogenic microbial specimens.<h4>Methodology/principal findings</h4>Silkworm fecal matter extracts prepared in 0.02 M phosphate buffer saline (pH 7.4), at a temperature of 60°C was subjected to 40% saturated ammonium sulphate precipitation and purified by gel-filtration chromatography (GFC). SDS-PAGE under denaturing conditions showed a single band at about 21.5 kDa. The peak fraction, thus obtained by GFC wastested for homogeneityusing C18reverse-phase high performance liquid chromatography (HPLC). The activity of the purified protein was tested against selected Gram +/- bacteria and phytopathogenic Fusarium species with concentration-dependent inhibitionrelationship. The purified bioactive protein was subjected to matrix-assisted laser desorption and ionization-time of flight mass spectrometry (MALDI-TOF-MS) and N-terminal sequencing by Edman degradation towards its identification. The N-terminal first 18 amino acid sequence following the predicted signal peptide showed homology to plant germin-like proteins (Glp). In order to characterize the full-length gene sequence in detail, the partial cDNA was cloned and sequenced using degenerate primers, followed by 5'- and 3'-rapid amplification of cDNA ends (RACE-PCR). The full-length cDNA sequence composed of 630 bp encoding 209 amino acids and corresponded to germin-like proteins (Glps) involved in plant development and defense.<h4>Conclusions/significance</h4>The study reports, characterization of novel Glpbelonging to subfamily 3 from M. alba by the purification of mature active protein from silkworm fecal matter. The N-terminal amino acid sequence of the purified protein was found similar to the deduced amino acid sequence (without the transit peptide sequence) of the full length cDNA from M. alba.https://www.ncbi.nlm.nih.gov/pmc/articles/pmid/23284650/?tool=EBI |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
Bharat Bhusan Patnaik Dong Hyun Kim Seung Han Oh Yong-Su Song Nguyen Dang Minh Chanh Jong Sun Kim Woo-jin Jung Atul Kumar Saha Bharat Bhushan Bindroo Yeon Soo Han |
spellingShingle |
Bharat Bhusan Patnaik Dong Hyun Kim Seung Han Oh Yong-Su Song Nguyen Dang Minh Chanh Jong Sun Kim Woo-jin Jung Atul Kumar Saha Bharat Bhushan Bindroo Yeon Soo Han Molecular cloning and characterization of novel Morus alba germin-like protein gene which encodes for a silkworm gut digestion-resistant antimicrobial protein. PLoS ONE |
author_facet |
Bharat Bhusan Patnaik Dong Hyun Kim Seung Han Oh Yong-Su Song Nguyen Dang Minh Chanh Jong Sun Kim Woo-jin Jung Atul Kumar Saha Bharat Bhushan Bindroo Yeon Soo Han |
author_sort |
Bharat Bhusan Patnaik |
title |
Molecular cloning and characterization of novel Morus alba germin-like protein gene which encodes for a silkworm gut digestion-resistant antimicrobial protein. |
title_short |
Molecular cloning and characterization of novel Morus alba germin-like protein gene which encodes for a silkworm gut digestion-resistant antimicrobial protein. |
title_full |
Molecular cloning and characterization of novel Morus alba germin-like protein gene which encodes for a silkworm gut digestion-resistant antimicrobial protein. |
title_fullStr |
Molecular cloning and characterization of novel Morus alba germin-like protein gene which encodes for a silkworm gut digestion-resistant antimicrobial protein. |
title_full_unstemmed |
Molecular cloning and characterization of novel Morus alba germin-like protein gene which encodes for a silkworm gut digestion-resistant antimicrobial protein. |
title_sort |
molecular cloning and characterization of novel morus alba germin-like protein gene which encodes for a silkworm gut digestion-resistant antimicrobial protein. |
publisher |
Public Library of Science (PLoS) |
series |
PLoS ONE |
issn |
1932-6203 |
publishDate |
2012-01-01 |
description |
<h4>Background</h4>Silkworm fecal matter is considered one of the richest sources of antimicrobial and antiviral protein (substances) and such economically feasible and eco-friendly proteins acting as secondary metabolites from the insect system can be explored for their practical utility in conferring broad spectrum disease resistance against pathogenic microbial specimens.<h4>Methodology/principal findings</h4>Silkworm fecal matter extracts prepared in 0.02 M phosphate buffer saline (pH 7.4), at a temperature of 60°C was subjected to 40% saturated ammonium sulphate precipitation and purified by gel-filtration chromatography (GFC). SDS-PAGE under denaturing conditions showed a single band at about 21.5 kDa. The peak fraction, thus obtained by GFC wastested for homogeneityusing C18reverse-phase high performance liquid chromatography (HPLC). The activity of the purified protein was tested against selected Gram +/- bacteria and phytopathogenic Fusarium species with concentration-dependent inhibitionrelationship. The purified bioactive protein was subjected to matrix-assisted laser desorption and ionization-time of flight mass spectrometry (MALDI-TOF-MS) and N-terminal sequencing by Edman degradation towards its identification. The N-terminal first 18 amino acid sequence following the predicted signal peptide showed homology to plant germin-like proteins (Glp). In order to characterize the full-length gene sequence in detail, the partial cDNA was cloned and sequenced using degenerate primers, followed by 5'- and 3'-rapid amplification of cDNA ends (RACE-PCR). The full-length cDNA sequence composed of 630 bp encoding 209 amino acids and corresponded to germin-like proteins (Glps) involved in plant development and defense.<h4>Conclusions/significance</h4>The study reports, characterization of novel Glpbelonging to subfamily 3 from M. alba by the purification of mature active protein from silkworm fecal matter. The N-terminal amino acid sequence of the purified protein was found similar to the deduced amino acid sequence (without the transit peptide sequence) of the full length cDNA from M. alba. |
url |
https://www.ncbi.nlm.nih.gov/pmc/articles/pmid/23284650/?tool=EBI |
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