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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Autores principales:	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
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2012
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/03e2469817f4457888f1201b2dcc7cb7
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spelling	oai:doaj.org-article:03e2469817f4457888f1201b2dcc7cb72021-11-18T08:04:35ZMolecular cloning and characterization of novel Morus alba germin-like protein gene which encodes for a silkworm gut digestion-resistant antimicrobial protein.1932-620310.1371/journal.pone.0050900https://doaj.org/article/03e2469817f4457888f1201b2dcc7cb72012-01-01T00:00:00Zhttps://www.ncbi.nlm.nih.gov/pmc/articles/pmid/23284650/?tool=EBIhttps://doaj.org/toc/1932-6203<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.Bharat Bhusan PatnaikDong Hyun KimSeung Han OhYong-Su SongNguyen Dang Minh ChanhJong Sun KimWoo-jin JungAtul Kumar SahaBharat Bhushan BindrooYeon Soo HanPublic Library of Science (PLoS)articleMedicineRScienceQENPLoS ONE, Vol 7, Iss 12, p e50900 (2012)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q 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.
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.
format	article
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
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)
publishDate	2012
url	https://doaj.org/article/03e2469817f4457888f1201b2dcc7cb7
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Molecular cloning and characterization of novel Morus alba germin-like protein gene which encodes for a silkworm gut digestion-resistant antimicrobial protein.

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