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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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Antifungal activity of purified novel protein.Relationship between inhibition probability and protein concentration logarithm for (A) F. solani and (B) F. oxysporum. The IR % of the purified protein against Fusarium species was converted to probit scale (Table 3). The probit of growth inhibition and log of protein concentration was plotted to calculate IC50 values from the correlation coefficients obtained.
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pone-0050900-g006: Antifungal activity of purified novel protein.Relationship between inhibition probability and protein concentration logarithm for (A) F. solani and (B) F. oxysporum. The IR % of the purified protein against Fusarium species was converted to probit scale (Table 3). The probit of growth inhibition and log of protein concentration was plotted to calculate IC50 values from the correlation coefficients obtained.

Mentions: The results of antifungal assays of the purified proteins as shown in Fig. 5 indicated high antifungal activity against fungal strains as F. solani and F. oxysporum. The observed activity against F. solani (Fig. 5A)was recorded at a protein concentration of 50 and 60 µg/µl, even though with the increase in incubation time the activity was reduced.. There was a significant increase in activity at 1 day incubation, followed with a significant decline at the 2nd day followed with stable maintenance of activity after 2 days. The pattern was similar with F. oxysporum (Fig. 5B), though the significant decline in activity was stabilized after 3 days of incubation with the purified protein. At a lower concentration of the protein, the activity was not marked with the increase in incubation time. The inhibitory concentration mean values were changed into the probit scale (Table 3) and were plotted against log of protein concentration to study the correlation and deduce the IC50 values (Fig. 6). The IC50 or effective concentration (EC50) values for F. solani and F. oxysporum was 56.8 and 58.43 µg/µl respectively. Antifungal assay for SE-RFP showed a good activity against C. albicans and A. flavus, whereas activity was low against A. niger as observed from the MIC values.


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)

Antifungal activity of purified novel protein.Relationship between inhibition probability and protein concentration logarithm for (A) F. solani and (B) F. oxysporum. The IR % of the purified protein against Fusarium species was converted to probit scale (Table 3). The probit of growth inhibition and log of protein concentration was plotted to calculate IC50 values from the correlation coefficients obtained.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0050900-g006: Antifungal activity of purified novel protein.Relationship between inhibition probability and protein concentration logarithm for (A) F. solani and (B) F. oxysporum. The IR % of the purified protein against Fusarium species was converted to probit scale (Table 3). The probit of growth inhibition and log of protein concentration was plotted to calculate IC50 values from the correlation coefficients obtained.
Mentions: The results of antifungal assays of the purified proteins as shown in Fig. 5 indicated high antifungal activity against fungal strains as F. solani and F. oxysporum. The observed activity against F. solani (Fig. 5A)was recorded at a protein concentration of 50 and 60 µg/µl, even though with the increase in incubation time the activity was reduced.. There was a significant increase in activity at 1 day incubation, followed with a significant decline at the 2nd day followed with stable maintenance of activity after 2 days. The pattern was similar with F. oxysporum (Fig. 5B), though the significant decline in activity was stabilized after 3 days of incubation with the purified protein. At a lower concentration of the protein, the activity was not marked with the increase in incubation time. The inhibitory concentration mean values were changed into the probit scale (Table 3) and were plotted against log of protein concentration to study the correlation and deduce the IC50 values (Fig. 6). The IC50 or effective concentration (EC50) values for F. solani and F. oxysporum was 56.8 and 58.43 µg/µl respectively. Antifungal assay for SE-RFP showed a good activity against C. albicans and A. flavus, whereas activity was low against A. niger as observed from the MIC values.

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