Limits...
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.

Show MeSH

Related in: MedlinePlus

Primary sequence analysis of novel germin-like protein from M. alba.(A) Multiple alignment of the deduced amino acid sequence of Ma-Glp with true germins and Glps from other representative plant groups as presented in Table 2. Residues shared by several germins and Glps are indicated with the following symbols: ‘*’-denotes identical residues in all sequences; ‘:’- conserved substitutions according to similar properties of amino acids; ‘.’- semi-conserved substitutions. Dashes indicate gaps used to maximize the alignment. (B) Relationships between members of several species presented as a phylogenetic tree andin ferred using the UPGMA method. The percentage of replicate trees in which the associated taxa clustered together in bootstrap test (5000 replicates) is shown next to branches. Evolutionary analysis was conducted in MEGA 5.0. Circular representation of tree. Cones- Glp subfamily III; Triangle-Glp subfamily II; Circle- Glp subfamily I; Square- True germins (C) Percent identity matrix of the representative species as inferred using Clustal X 1.83.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3526618&req=5

pone-0050900-g013: Primary sequence analysis of novel germin-like protein from M. alba.(A) Multiple alignment of the deduced amino acid sequence of Ma-Glp with true germins and Glps from other representative plant groups as presented in Table 2. Residues shared by several germins and Glps are indicated with the following symbols: ‘*’-denotes identical residues in all sequences; ‘:’- conserved substitutions according to similar properties of amino acids; ‘.’- semi-conserved substitutions. Dashes indicate gaps used to maximize the alignment. (B) Relationships between members of several species presented as a phylogenetic tree andin ferred using the UPGMA method. The percentage of replicate trees in which the associated taxa clustered together in bootstrap test (5000 replicates) is shown next to branches. Evolutionary analysis was conducted in MEGA 5.0. Circular representation of tree. Cones- Glp subfamily III; Triangle-Glp subfamily II; Circle- Glp subfamily I; Square- True germins (C) Percent identity matrix of the representative species as inferred using Clustal X 1.83.

Mentions: The full-length sequence of Ma-Glp was found to comprise of 1238 nucleotides with a cDNA sequence of 630 bp. The cDNA encodes a protein of 209 amino acid residues. The 5′-, 3′- non-coding and poly (A+) sequences were 93, 515 and 27 bp, respectively (Fig. 12). Multiple alignments of Ma-GLP sequences with other reported true germin/Glp genes (Fig. 13A) highlighted number of conserved motifs and structural similarities that are common to the plant Glp subfamily [44]. The deduced amino acid sequence of Ma-Glp comprised of a conserved extracellular targeting signal peptide (Fig. 14) located at the N-terminus that is a characteristic feature of the Glp gene family with the exception of Arachis hypogea Glp7, predicted to contain a non-cleavable amino-terminal sequence. The lack of a KDEL consensus motif in the sequence targets the protein into the secretory pathway rather than endoplasmic reticulum retention. The signal peptide is predicted to be cleaved between amino acid residues 18 and 19 and the 19th residue acts as the first residue of the mature secreted protein [45], [46]. The mature protein without the putative signal peptide region was found to encode 191 amino acid residues. It is therefore significant here to point out that the bioactive protein purified from silkworm fecal resource is the mature protein or the secreted product of the food plant, M. alba.


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)

Primary sequence analysis of novel germin-like protein from M. alba.(A) Multiple alignment of the deduced amino acid sequence of Ma-Glp with true germins and Glps from other representative plant groups as presented in Table 2. Residues shared by several germins and Glps are indicated with the following symbols: ‘*’-denotes identical residues in all sequences; ‘:’- conserved substitutions according to similar properties of amino acids; ‘.’- semi-conserved substitutions. Dashes indicate gaps used to maximize the alignment. (B) Relationships between members of several species presented as a phylogenetic tree andin ferred using the UPGMA method. The percentage of replicate trees in which the associated taxa clustered together in bootstrap test (5000 replicates) is shown next to branches. Evolutionary analysis was conducted in MEGA 5.0. Circular representation of tree. Cones- Glp subfamily III; Triangle-Glp subfamily II; Circle- Glp subfamily I; Square- True germins (C) Percent identity matrix of the representative species as inferred using Clustal X 1.83.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0050900-g013: Primary sequence analysis of novel germin-like protein from M. alba.(A) Multiple alignment of the deduced amino acid sequence of Ma-Glp with true germins and Glps from other representative plant groups as presented in Table 2. Residues shared by several germins and Glps are indicated with the following symbols: ‘*’-denotes identical residues in all sequences; ‘:’- conserved substitutions according to similar properties of amino acids; ‘.’- semi-conserved substitutions. Dashes indicate gaps used to maximize the alignment. (B) Relationships between members of several species presented as a phylogenetic tree andin ferred using the UPGMA method. The percentage of replicate trees in which the associated taxa clustered together in bootstrap test (5000 replicates) is shown next to branches. Evolutionary analysis was conducted in MEGA 5.0. Circular representation of tree. Cones- Glp subfamily III; Triangle-Glp subfamily II; Circle- Glp subfamily I; Square- True germins (C) Percent identity matrix of the representative species as inferred using Clustal X 1.83.
Mentions: The full-length sequence of Ma-Glp was found to comprise of 1238 nucleotides with a cDNA sequence of 630 bp. The cDNA encodes a protein of 209 amino acid residues. The 5′-, 3′- non-coding and poly (A+) sequences were 93, 515 and 27 bp, respectively (Fig. 12). Multiple alignments of Ma-GLP sequences with other reported true germin/Glp genes (Fig. 13A) highlighted number of conserved motifs and structural similarities that are common to the plant Glp subfamily [44]. The deduced amino acid sequence of Ma-Glp comprised of a conserved extracellular targeting signal peptide (Fig. 14) located at the N-terminus that is a characteristic feature of the Glp gene family with the exception of Arachis hypogea Glp7, predicted to contain a non-cleavable amino-terminal sequence. The lack of a KDEL consensus motif in the sequence targets the protein into the secretory pathway rather than endoplasmic reticulum retention. The signal peptide is predicted to be cleaved between amino acid residues 18 and 19 and the 19th residue acts as the first residue of the mature secreted protein [45], [46]. The mature protein without the putative signal peptide region was found to encode 191 amino acid residues. It is therefore significant here to point out that the bioactive protein purified from silkworm fecal resource is the mature protein or the secreted product of the food plant, M. alba.

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