Limits...
Isolation and preliminary characterization of proteinaceous toxins with insecticidal and antibacterial activities from black widow spider (L. tredecimguttatus) eggs.

Lei Q, Yu H, Peng X, Yan S, Wang J, Yan Y, Wang X - Toxins (Basel) (2015)

Bottom Line: Latroeggtoxin-III was demonstrated to have a molecular weight of about 36 kDa.Activity analysis indicated that latroeggtoxin-III exhibited neurotoxicity against cockroaches but had no obvious effect on mice, suggesting that it is an insect-specific toxin.Finally, the implications of the proteinaceous toxins in egg protection and their potential applications were analyzed and discussed.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory of Protein Chemistry and Developmental Biology of Ministry of Education, College of Life Sciences, Hunan Normal University, Changsha 410081, China. leiqian0607@163.com.

ABSTRACT
The eggs of black widow spider (L. tredecimguttatus) have been demonstrated to be rich in toxic proteinaceous components. The study on such active components is of theoretical and practical importance. In the present work, using a combination of multiple biochemical and biological strategies, we isolated and characterized the proteinaceous components from the aqueous extract of the black widow spider eggs. After gel filtration of the egg extract, the resulting main protein and peptide peaks were further fractionated by ion exchange chromatography and reversed-phase high performance liquid chromatography. Two proteinaceous components, named latroeggtoxin-III and latroeggtoxin-IV, respectively, were purified to homogeneity. Latroeggtoxin-III was demonstrated to have a molecular weight of about 36 kDa. Activity analysis indicated that latroeggtoxin-III exhibited neurotoxicity against cockroaches but had no obvious effect on mice, suggesting that it is an insect-specific toxin. Latroeggtoxin-IV, with a molecular weight of 3.6 kDa, was shown to be a broad-spectrum antibacterial peptide, showing inhibitory activity against all five species of bacteria tested, with the highest activity against Staphylococcus aureus. Finally, the implications of the proteinaceous toxins in egg protection and their potential applications were analyzed and discussed.

Show MeSH

Related in: MedlinePlus

Further separation of the fraction F1 from molecular sieve chromatography. (A) Anion exchange chromatography of the fraction F1; F1 was the first fraction obtained from molecular sieve chromatography in Figure 1 and was further separated into four peaks, named F1-1 to F1-4; (B) RP-HPLC of the F1-3 from anion exchange chromatography of the fraction F1. The peak indicated with an arrow was Latroeggtoxin-III.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4379531&req=5

toxins-07-00886-f002: Further separation of the fraction F1 from molecular sieve chromatography. (A) Anion exchange chromatography of the fraction F1; F1 was the first fraction obtained from molecular sieve chromatography in Figure 1 and was further separated into four peaks, named F1-1 to F1-4; (B) RP-HPLC of the F1-3 from anion exchange chromatography of the fraction F1. The peak indicated with an arrow was Latroeggtoxin-III.

Mentions: For further separation of the fraction F1, anion exchange and reversed-phase chromatographies were employed sequentially. A representative elution profile of anion exchange chromatography is shown in Figure 2A. From the figure it can be seen that the components in the fraction F1 were fractionated into four peaks (F1-1 to F1-4) and the complexity of the sample was further decreased. The main peak F1-3 was collected and was further separated with RP-HPLC. In view of the fact that the proteins to be isolated have a high molecular weight, we used a low-hydrophobicity C4 column in order to minimize the risk of protein denaturation. The representative RP-HPLC chromatogram is shown in Figure 2B. The peak indicated with an arrow was collected, which, together with egg extract and the partially purified samples, was analyzed by SDS-PAGE (Figure 3) to show purification efficiency in comparison. The results in the figure indicated that a combination of different chromatographic techniques such as molecular sieve filtration, ion-exchange and RP-HPLC could efficiently separate the proteins in the egg extract. During the purification processes the sample complexity was gradually decreased, and as a result a protein with a molecular weight of about 36 kDa was purified. The protein was named latroeggtoxin-III, because in previous work we had purified and characterized two different proteins (named Latroeggtoxin-I and Latroeggtoxin-II, respectively) from the black widow spider eggs [10,11].


Isolation and preliminary characterization of proteinaceous toxins with insecticidal and antibacterial activities from black widow spider (L. tredecimguttatus) eggs.

Lei Q, Yu H, Peng X, Yan S, Wang J, Yan Y, Wang X - Toxins (Basel) (2015)

Further separation of the fraction F1 from molecular sieve chromatography. (A) Anion exchange chromatography of the fraction F1; F1 was the first fraction obtained from molecular sieve chromatography in Figure 1 and was further separated into four peaks, named F1-1 to F1-4; (B) RP-HPLC of the F1-3 from anion exchange chromatography of the fraction F1. The peak indicated with an arrow was Latroeggtoxin-III.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4379531&req=5

toxins-07-00886-f002: Further separation of the fraction F1 from molecular sieve chromatography. (A) Anion exchange chromatography of the fraction F1; F1 was the first fraction obtained from molecular sieve chromatography in Figure 1 and was further separated into four peaks, named F1-1 to F1-4; (B) RP-HPLC of the F1-3 from anion exchange chromatography of the fraction F1. The peak indicated with an arrow was Latroeggtoxin-III.
Mentions: For further separation of the fraction F1, anion exchange and reversed-phase chromatographies were employed sequentially. A representative elution profile of anion exchange chromatography is shown in Figure 2A. From the figure it can be seen that the components in the fraction F1 were fractionated into four peaks (F1-1 to F1-4) and the complexity of the sample was further decreased. The main peak F1-3 was collected and was further separated with RP-HPLC. In view of the fact that the proteins to be isolated have a high molecular weight, we used a low-hydrophobicity C4 column in order to minimize the risk of protein denaturation. The representative RP-HPLC chromatogram is shown in Figure 2B. The peak indicated with an arrow was collected, which, together with egg extract and the partially purified samples, was analyzed by SDS-PAGE (Figure 3) to show purification efficiency in comparison. The results in the figure indicated that a combination of different chromatographic techniques such as molecular sieve filtration, ion-exchange and RP-HPLC could efficiently separate the proteins in the egg extract. During the purification processes the sample complexity was gradually decreased, and as a result a protein with a molecular weight of about 36 kDa was purified. The protein was named latroeggtoxin-III, because in previous work we had purified and characterized two different proteins (named Latroeggtoxin-I and Latroeggtoxin-II, respectively) from the black widow spider eggs [10,11].

Bottom Line: Latroeggtoxin-III was demonstrated to have a molecular weight of about 36 kDa.Activity analysis indicated that latroeggtoxin-III exhibited neurotoxicity against cockroaches but had no obvious effect on mice, suggesting that it is an insect-specific toxin.Finally, the implications of the proteinaceous toxins in egg protection and their potential applications were analyzed and discussed.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory of Protein Chemistry and Developmental Biology of Ministry of Education, College of Life Sciences, Hunan Normal University, Changsha 410081, China. leiqian0607@163.com.

ABSTRACT
The eggs of black widow spider (L. tredecimguttatus) have been demonstrated to be rich in toxic proteinaceous components. The study on such active components is of theoretical and practical importance. In the present work, using a combination of multiple biochemical and biological strategies, we isolated and characterized the proteinaceous components from the aqueous extract of the black widow spider eggs. After gel filtration of the egg extract, the resulting main protein and peptide peaks were further fractionated by ion exchange chromatography and reversed-phase high performance liquid chromatography. Two proteinaceous components, named latroeggtoxin-III and latroeggtoxin-IV, respectively, were purified to homogeneity. Latroeggtoxin-III was demonstrated to have a molecular weight of about 36 kDa. Activity analysis indicated that latroeggtoxin-III exhibited neurotoxicity against cockroaches but had no obvious effect on mice, suggesting that it is an insect-specific toxin. Latroeggtoxin-IV, with a molecular weight of 3.6 kDa, was shown to be a broad-spectrum antibacterial peptide, showing inhibitory activity against all five species of bacteria tested, with the highest activity against Staphylococcus aureus. Finally, the implications of the proteinaceous toxins in egg protection and their potential applications were analyzed and discussed.

Show MeSH
Related in: MedlinePlus