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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.

Patnaik BB, Kim DH, Oh SH, Song YS, Chanh ND, Kim JS, Jung WJ, Saha AK, Bindroo BB, Han YS - PLoS ONE (2012)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Division of Plant Biotechnology, College of Agriculture and Life Sciences, Chonnam National University, Gwangju, South Korea. drbharatbhusan4@gmail.com

ABSTRACT

Background: 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.

Methodology/principal findings: 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.

Conclusions/significance: 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.

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MALDI-TOF MS analysis of the purified novel protein.(A) Determination of intact molecular mass using Sinapinic acid as matrix. Protein samples were cleaned using the ZipTip ™ C18 resin. The scan range between 10,000–80,000 Da is presented with the singly charged peak [M+H]+for the target protein is depicted by arrow. (B) In-gel tryptic digestion spectra of the protein using α-cyano-4-hydroxycinnamic acid (CHCA) as matrix. The scan range between 800–4000 Da is presented with the appropriate masses of generated peptides is highlighted on the top of the peak. In-gel tryptic map of BSA in CHCA matrix was used as the internal calibrated standard.
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pone-0050900-g008: MALDI-TOF MS analysis of the purified novel protein.(A) Determination of intact molecular mass using Sinapinic acid as matrix. Protein samples were cleaned using the ZipTip ™ C18 resin. The scan range between 10,000–80,000 Da is presented with the singly charged peak [M+H]+for the target protein is depicted by arrow. (B) In-gel tryptic digestion spectra of the protein using α-cyano-4-hydroxycinnamic acid (CHCA) as matrix. The scan range between 800–4000 Da is presented with the appropriate masses of generated peptides is highlighted on the top of the peak. In-gel tryptic map of BSA in CHCA matrix was used as the internal calibrated standard.

Mentions: The completely purified protein from silkworm fecal extract was subjected to MALDI-TOF-MS for determination of intact molecular mass and subsequently the in-gel separated proteins were digested with trypsin to generate the peptide fragments towards protein identification. MALDI-TOF-MS analysis of purified protein showed a molecular mass of 21,285 Da (Fig. 8A), which was in good agreement with the size observed by SDS-PAGE. The m/z values for in-gel digested peptide fragments from the purified protein was generated using MALDI-TOF-MS (Fig. 8B) and was queried for significant match using the available public databases. The mass of the peptide fragments generated did not conform to any significant matches within the database. It is assumed here that the covalent modifications of the polypeptides in the insect gut may prevent protein identification by MS. This was significant towards progress of our attempts to characterize and identify the protein by Edman sequencing.


Molecular cloning and characterization of novel Morus alba germin-like protein gene which encodes for a silkworm gut digestion-resistant antimicrobial protein.

Patnaik BB, Kim DH, Oh SH, Song YS, Chanh ND, Kim JS, Jung WJ, Saha AK, Bindroo BB, Han YS - PLoS ONE (2012)

MALDI-TOF MS analysis of the purified novel protein.(A) Determination of intact molecular mass using Sinapinic acid as matrix. Protein samples were cleaned using the ZipTip ™ C18 resin. The scan range between 10,000–80,000 Da is presented with the singly charged peak [M+H]+for the target protein is depicted by arrow. (B) In-gel tryptic digestion spectra of the protein using α-cyano-4-hydroxycinnamic acid (CHCA) as matrix. The scan range between 800–4000 Da is presented with the appropriate masses of generated peptides is highlighted on the top of the peak. In-gel tryptic map of BSA in CHCA matrix was used as the internal calibrated standard.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3526618&req=5

pone-0050900-g008: MALDI-TOF MS analysis of the purified novel protein.(A) Determination of intact molecular mass using Sinapinic acid as matrix. Protein samples were cleaned using the ZipTip ™ C18 resin. The scan range between 10,000–80,000 Da is presented with the singly charged peak [M+H]+for the target protein is depicted by arrow. (B) In-gel tryptic digestion spectra of the protein using α-cyano-4-hydroxycinnamic acid (CHCA) as matrix. The scan range between 800–4000 Da is presented with the appropriate masses of generated peptides is highlighted on the top of the peak. In-gel tryptic map of BSA in CHCA matrix was used as the internal calibrated standard.
Mentions: The completely purified protein from silkworm fecal extract was subjected to MALDI-TOF-MS for determination of intact molecular mass and subsequently the in-gel separated proteins were digested with trypsin to generate the peptide fragments towards protein identification. MALDI-TOF-MS analysis of purified protein showed a molecular mass of 21,285 Da (Fig. 8A), which was in good agreement with the size observed by SDS-PAGE. The m/z values for in-gel digested peptide fragments from the purified protein was generated using MALDI-TOF-MS (Fig. 8B) and was queried for significant match using the available public databases. The mass of the peptide fragments generated did not conform to any significant matches within the database. It is assumed here that the covalent modifications of the polypeptides in the insect gut may prevent protein identification by MS. This was significant towards progress of our attempts to characterize and identify the protein by Edman sequencing.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Division of Plant Biotechnology, College of Agriculture and Life Sciences, Chonnam National University, Gwangju, South Korea. drbharatbhusan4@gmail.com

ABSTRACT

Background: 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.

Methodology/principal findings: 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.

Conclusions/significance: 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.

Show MeSH
Related in: MedlinePlus