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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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Related in: MedlinePlus

Gel filtration chromatography profile of bioactive peak ‘A’ fraction.The column purified dialysate was subjected to Gel filtration chromatography using Sephadex G-75 column (1.6×70 cm) at a flow rate of 0.1 ml/min and sample volume of 500 µl. Protein was eluted with 0.02 M phosphate buffer solution at pH 7.4. The retention time of the peak was 483.2 min with an area of 899.6 mAU*min and a peak area of 90.5%.
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pone-0050900-g001: Gel filtration chromatography profile of bioactive peak ‘A’ fraction.The column purified dialysate was subjected to Gel filtration chromatography using Sephadex G-75 column (1.6×70 cm) at a flow rate of 0.1 ml/min and sample volume of 500 µl. Protein was eluted with 0.02 M phosphate buffer solution at pH 7.4. The retention time of the peak was 483.2 min with an area of 899.6 mAU*min and a peak area of 90.5%.

Mentions: The silkworm fecal matter is one of the rich and promising sources for exploring proteins having potent antimicrobial and antiviral functions. The present study explored the thermally-resistant bioactive protein components of silkworm fecal resources as the extraction process was accomplished at an elevated temperature of 60°C. Novel bioactive proteins were subsequently purified by conventional biochemical techniques, such as 40% ammonium sulphate precipitation, silica column chromatography and GFC with Sephadex G-75. GFC with Sephadex G-75 resin as matrix resolved the column chromatography purified fractions into two peaks A and B. Peak A was resolved as a sharper peak, whereas peak B was observed as a broader peak and assumed to contain low molecular weight proteins/peptides. The UV spectrum plot was recorded at absorption of 280 nm. The peak A fraction had a retention time and volume of 483 min and 43 ml. respectively as observed in AKTA Prime Unicorn 5.0 software (Fig. 1). Subsequently, the peak A fractions were characterized by denaturing and non-denaturing gel systems and tested for its positive activity against some pathogenic microbes (bacteria and fungi).To check the homogeneity of the bioactive peak fraction obtained from GFC, the above sample was lyophilized and subjected for HPLC using reverse- phase C18 column. A prominent peak (Fig. 2) at a retention time of 12.95 min. was observed that was purified using preparatory column for HPLC. Analytical-scale HPLC is always the final step to check for the purity of the already prepared and characterized proteins and have been consistently used in most of the earlier referred reports.


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)

Gel filtration chromatography profile of bioactive peak ‘A’ fraction.The column purified dialysate was subjected to Gel filtration chromatography using Sephadex G-75 column (1.6×70 cm) at a flow rate of 0.1 ml/min and sample volume of 500 µl. Protein was eluted with 0.02 M phosphate buffer solution at pH 7.4. The retention time of the peak was 483.2 min with an area of 899.6 mAU*min and a peak area of 90.5%.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0050900-g001: Gel filtration chromatography profile of bioactive peak ‘A’ fraction.The column purified dialysate was subjected to Gel filtration chromatography using Sephadex G-75 column (1.6×70 cm) at a flow rate of 0.1 ml/min and sample volume of 500 µl. Protein was eluted with 0.02 M phosphate buffer solution at pH 7.4. The retention time of the peak was 483.2 min with an area of 899.6 mAU*min and a peak area of 90.5%.
Mentions: The silkworm fecal matter is one of the rich and promising sources for exploring proteins having potent antimicrobial and antiviral functions. The present study explored the thermally-resistant bioactive protein components of silkworm fecal resources as the extraction process was accomplished at an elevated temperature of 60°C. Novel bioactive proteins were subsequently purified by conventional biochemical techniques, such as 40% ammonium sulphate precipitation, silica column chromatography and GFC with Sephadex G-75. GFC with Sephadex G-75 resin as matrix resolved the column chromatography purified fractions into two peaks A and B. Peak A was resolved as a sharper peak, whereas peak B was observed as a broader peak and assumed to contain low molecular weight proteins/peptides. The UV spectrum plot was recorded at absorption of 280 nm. The peak A fraction had a retention time and volume of 483 min and 43 ml. respectively as observed in AKTA Prime Unicorn 5.0 software (Fig. 1). Subsequently, the peak A fractions were characterized by denaturing and non-denaturing gel systems and tested for its positive activity against some pathogenic microbes (bacteria and fungi).To check the homogeneity of the bioactive peak fraction obtained from GFC, the above sample was lyophilized and subjected for HPLC using reverse- phase C18 column. A prominent peak (Fig. 2) at a retention time of 12.95 min. was observed that was purified using preparatory column for HPLC. Analytical-scale HPLC is always the final step to check for the purity of the already prepared and characterized proteins and have been consistently used in most of the earlier referred reports.

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