Purification and Gene Cloning of a Novel Antibacterial Phospholipase A2 from the Sponge Agelas Clathroides In Kapoposang Island Indonesia Terrestrial

Ahyar Ahmad; Hasnah Natsir; Harningsih Karim

doi:doi:10.11648/j.ajbls.20140205.13

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Purification and Gene Cloning of a Novel Antibacterial Phospholipase A2 from the Sponge Agelas Clathroides In Kapoposang Island Indonesia Terrestrial

Ahyar Ahmad, Hasnah Natsir, Harningsih Karim

Published in American Journal of Biomedical and Life Sciences (Volume 2, Issue 5)

Received: 6 October 2014 Accepted: 16 October 2014 Published: 30 October 2014

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Abstract

A new phospholipase A2 enzyme (PLA2) has been purified from the sponge of Agelas clathroides by using ammonium sulphate fractionation, column chromatography and reversed-phase HPLC. It behaves as a single-band on SDS-PAGE with molecular weight of 39 kDa. Based on amino acids partial sequence, we cloned and sequenced cDNA encoding PLA2. It consists of 474 nucleotides encoding 157 amino acid residues including a putative initiation Met. To obtain it in large amounts, the coding sequence of PLA2 was cloned into pGEX-2TK vector and expressed as a PLA2 fusion protein in Escherichia coli BL21 strain. The soluble fusion protein collected from the supernatant of the cell lysate with induction by 50 M isopropyl-β-D-thiogalactopyranoside (IPTG) was purified in a single-step on glutathione agarose bead chromatography. The purified native PLA2 protein and recombinant PLA2 fusion protein were determinated for novel antibacterial activity. Recombinant PLA2 fusion protein exhibited a similar antibacterial activity to the native PLA2. The recombinant PLA2 had stronger antibacterial activity toward Salmonella typhy and Staphylococus aureus (G+) with the inhibition zone diameters of 2.0 times higher than that toward Echerichia coli and Vibrio cholerae (G-). These works might provide a significant foundation for following research on the antibacterial action of PLA2 protein from marine sponges.

Published in	American Journal of Biomedical and Life Sciences (Volume 2, Issue 5)
DOI	10.11648/j.ajbls.20140205.13
Page(s)	119-126
Creative Commons	This is an Open Access article, distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution and reproduction in any medium or format, provided the original work is properly cited.
Copyright	Copyright © The Author(s), 2014. Published by Science Publishing Group

Keywords

Sponge, Agelas clathroides, Phospholipase A2, Recombinant Protein, Antibacterial Activity

References

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APA Style

Ahyar Ahmad, Hasnah Natsir, Harningsih Karim. (2014). Purification and Gene Cloning of a Novel Antibacterial Phospholipase A2 from the Sponge Agelas Clathroides In Kapoposang Island Indonesia Terrestrial. American Journal of Biomedical and Life Sciences, 2(5), 119-126. https://doi.org/10.11648/j.ajbls.20140205.13

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ACS Style

Ahyar Ahmad; Hasnah Natsir; Harningsih Karim. Purification and Gene Cloning of a Novel Antibacterial Phospholipase A2 from the Sponge Agelas Clathroides In Kapoposang Island Indonesia Terrestrial. Am. J. Biomed. Life Sci. 2014, 2(5), 119-126. doi: 10.11648/j.ajbls.20140205.13

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AMA Style

Ahyar Ahmad, Hasnah Natsir, Harningsih Karim. Purification and Gene Cloning of a Novel Antibacterial Phospholipase A2 from the Sponge Agelas Clathroides In Kapoposang Island Indonesia Terrestrial. Am J Biomed Life Sci. 2014;2(5):119-126. doi: 10.11648/j.ajbls.20140205.13

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@article{10.11648/j.ajbls.20140205.13,
  author = {Ahyar Ahmad and Hasnah Natsir and Harningsih Karim},
  title = {Purification and Gene Cloning of a Novel Antibacterial Phospholipase A2 from the Sponge Agelas Clathroides In Kapoposang Island Indonesia Terrestrial},
  journal = {American Journal of Biomedical and Life Sciences},
  volume = {2},
  number = {5},
  pages = {119-126},
  doi = {10.11648/j.ajbls.20140205.13},
  url = {https://doi.org/10.11648/j.ajbls.20140205.13},
  eprint = {https://article.sciencepublishinggroup.com/pdf/10.11648.j.ajbls.20140205.13},
  abstract = {A new phospholipase A2 enzyme (PLA2) has been purified from the sponge of Agelas clathroides by using ammonium sulphate fractionation, column chromatography and reversed-phase HPLC. It behaves as a single-band on SDS-PAGE with molecular weight of 39 kDa. Based on amino acids partial sequence, we cloned and sequenced cDNA encoding PLA2. It consists of 474 nucleotides encoding 157 amino acid residues including a putative initiation Met. To obtain it in large amounts, the coding sequence of PLA2 was cloned into pGEX-2TK vector and expressed as a PLA2 fusion protein in Escherichia coli BL21 strain. The soluble fusion protein collected from the supernatant of the cell lysate with induction by 50 M isopropyl-β-D-thiogalactopyranoside (IPTG) was purified in a single-step on glutathione agarose bead chromatography. The purified native PLA2 protein and recombinant PLA2 fusion protein were determinated for novel antibacterial activity. Recombinant PLA2 fusion protein exhibited a similar antibacterial activity to the native PLA2. The recombinant PLA2 had stronger antibacterial activity toward Salmonella typhy and Staphylococus aureus (G+) with the inhibition zone diameters of 2.0 times higher than that toward Echerichia coli and Vibrio cholerae (G-). These works might provide a significant foundation for following research on the antibacterial action of PLA2 protein from marine sponges.},
 year = {2014}
}

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TY  - JOUR
T1  - Purification and Gene Cloning of a Novel Antibacterial Phospholipase A2 from the Sponge Agelas Clathroides In Kapoposang Island Indonesia Terrestrial
AU  - Ahyar Ahmad
AU  - Hasnah Natsir
AU  - Harningsih Karim
Y1  - 2014/10/30
PY  - 2014
N1  - https://doi.org/10.11648/j.ajbls.20140205.13
DO  - 10.11648/j.ajbls.20140205.13
T2  - American Journal of Biomedical and Life Sciences
JF  - American Journal of Biomedical and Life Sciences
JO  - American Journal of Biomedical and Life Sciences
SP  - 119
EP  - 126
PB  - Science Publishing Group
SN  - 2330-880X
UR  - https://doi.org/10.11648/j.ajbls.20140205.13
AB  - A new phospholipase A2 enzyme (PLA2) has been purified from the sponge of Agelas clathroides by using ammonium sulphate fractionation, column chromatography and reversed-phase HPLC. It behaves as a single-band on SDS-PAGE with molecular weight of 39 kDa. Based on amino acids partial sequence, we cloned and sequenced cDNA encoding PLA2. It consists of 474 nucleotides encoding 157 amino acid residues including a putative initiation Met. To obtain it in large amounts, the coding sequence of PLA2 was cloned into pGEX-2TK vector and expressed as a PLA2 fusion protein in Escherichia coli BL21 strain. The soluble fusion protein collected from the supernatant of the cell lysate with induction by 50 M isopropyl-β-D-thiogalactopyranoside (IPTG) was purified in a single-step on glutathione agarose bead chromatography. The purified native PLA2 protein and recombinant PLA2 fusion protein were determinated for novel antibacterial activity. Recombinant PLA2 fusion protein exhibited a similar antibacterial activity to the native PLA2. The recombinant PLA2 had stronger antibacterial activity toward Salmonella typhy and Staphylococus aureus (G+) with the inhibition zone diameters of 2.0 times higher than that toward Echerichia coli and Vibrio cholerae (G-). These works might provide a significant foundation for following research on the antibacterial action of PLA2 protein from marine sponges.
VL  - 2
IS  - 5
ER  -

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Author Information

Ahyar Ahmad

Laboratory of Research Centre and Developing of Sciences, Mathematic and Natural Science Faculty, Hasanuddin University, Jl. Perintis Kemerdekaan KM 10, Makassar 90245, Indonesia
Hasnah Natsir

Laboratory of Biochemistry, Chemistry Department, Mathematic and Natural Science Faculty, Hasanuddin Univesity, Jl. Perintis Kemerdekaan KM 10, Makassar 90245, Indonesia
Harningsih Karim

Department of Pharmacy, School of Pharmacy YAMASI, Jl. Mapala 2 Blok D5 No.10 Makassar 90222, Indonesia

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Ahyar Ahmad, Hasnah Natsir, Harningsih Karim. (2014). Purification and Gene Cloning of a Novel Antibacterial Phospholipase A2 from the Sponge Agelas Clathroides In Kapoposang Island Indonesia Terrestrial. American Journal of Biomedical and Life Sciences, 2(5), 119-126. https://doi.org/10.11648/j.ajbls.20140205.13

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ACS Style

Ahyar Ahmad; Hasnah Natsir; Harningsih Karim. Purification and Gene Cloning of a Novel Antibacterial Phospholipase A2 from the Sponge Agelas Clathroides In Kapoposang Island Indonesia Terrestrial. Am. J. Biomed. Life Sci. 2014, 2(5), 119-126. doi: 10.11648/j.ajbls.20140205.13

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AMA Style

Ahyar Ahmad, Hasnah Natsir, Harningsih Karim. Purification and Gene Cloning of a Novel Antibacterial Phospholipase A2 from the Sponge Agelas Clathroides In Kapoposang Island Indonesia Terrestrial. Am J Biomed Life Sci. 2014;2(5):119-126. doi: 10.11648/j.ajbls.20140205.13

Copy | Download

@article{10.11648/j.ajbls.20140205.13,
  author = {Ahyar Ahmad and Hasnah Natsir and Harningsih Karim},
  title = {Purification and Gene Cloning of a Novel Antibacterial Phospholipase A2 from the Sponge Agelas Clathroides In Kapoposang Island Indonesia Terrestrial},
  journal = {American Journal of Biomedical and Life Sciences},
  volume = {2},
  number = {5},
  pages = {119-126},
  doi = {10.11648/j.ajbls.20140205.13},
  url = {https://doi.org/10.11648/j.ajbls.20140205.13},
  eprint = {https://article.sciencepublishinggroup.com/pdf/10.11648.j.ajbls.20140205.13},
  abstract = {A new phospholipase A2 enzyme (PLA2) has been purified from the sponge of Agelas clathroides by using ammonium sulphate fractionation, column chromatography and reversed-phase HPLC. It behaves as a single-band on SDS-PAGE with molecular weight of 39 kDa. Based on amino acids partial sequence, we cloned and sequenced cDNA encoding PLA2. It consists of 474 nucleotides encoding 157 amino acid residues including a putative initiation Met. To obtain it in large amounts, the coding sequence of PLA2 was cloned into pGEX-2TK vector and expressed as a PLA2 fusion protein in Escherichia coli BL21 strain. The soluble fusion protein collected from the supernatant of the cell lysate with induction by 50 M isopropyl-β-D-thiogalactopyranoside (IPTG) was purified in a single-step on glutathione agarose bead chromatography. The purified native PLA2 protein and recombinant PLA2 fusion protein were determinated for novel antibacterial activity. Recombinant PLA2 fusion protein exhibited a similar antibacterial activity to the native PLA2. The recombinant PLA2 had stronger antibacterial activity toward Salmonella typhy and Staphylococus aureus (G+) with the inhibition zone diameters of 2.0 times higher than that toward Echerichia coli and Vibrio cholerae (G-). These works might provide a significant foundation for following research on the antibacterial action of PLA2 protein from marine sponges.},
 year = {2014}
}

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TY  - JOUR
T1  - Purification and Gene Cloning of a Novel Antibacterial Phospholipase A2 from the Sponge Agelas Clathroides In Kapoposang Island Indonesia Terrestrial
AU  - Ahyar Ahmad
AU  - Hasnah Natsir
AU  - Harningsih Karim
Y1  - 2014/10/30
PY  - 2014
N1  - https://doi.org/10.11648/j.ajbls.20140205.13
DO  - 10.11648/j.ajbls.20140205.13
T2  - American Journal of Biomedical and Life Sciences
JF  - American Journal of Biomedical and Life Sciences
JO  - American Journal of Biomedical and Life Sciences
SP  - 119
EP  - 126
PB  - Science Publishing Group
SN  - 2330-880X
UR  - https://doi.org/10.11648/j.ajbls.20140205.13
AB  - A new phospholipase A2 enzyme (PLA2) has been purified from the sponge of Agelas clathroides by using ammonium sulphate fractionation, column chromatography and reversed-phase HPLC. It behaves as a single-band on SDS-PAGE with molecular weight of 39 kDa. Based on amino acids partial sequence, we cloned and sequenced cDNA encoding PLA2. It consists of 474 nucleotides encoding 157 amino acid residues including a putative initiation Met. To obtain it in large amounts, the coding sequence of PLA2 was cloned into pGEX-2TK vector and expressed as a PLA2 fusion protein in Escherichia coli BL21 strain. The soluble fusion protein collected from the supernatant of the cell lysate with induction by 50 M isopropyl-β-D-thiogalactopyranoside (IPTG) was purified in a single-step on glutathione agarose bead chromatography. The purified native PLA2 protein and recombinant PLA2 fusion protein were determinated for novel antibacterial activity. Recombinant PLA2 fusion protein exhibited a similar antibacterial activity to the native PLA2. The recombinant PLA2 had stronger antibacterial activity toward Salmonella typhy and Staphylococus aureus (G+) with the inhibition zone diameters of 2.0 times higher than that toward Echerichia coli and Vibrio cholerae (G-). These works might provide a significant foundation for following research on the antibacterial action of PLA2 protein from marine sponges.
VL  - 2
IS  - 5
ER  -

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