Effect of a recombinant Lys49PLA2 myotoxin and Lys49PLA2-derived synthetic peptides from Agkistrodon species on membrane permeability to water

Aggregation is a serious obstacle for recovery of biologically active heterologous proteins from inclusion bodies (IBs) produced by recombinant bacteria. E. coli transformed with a vector containing the cDNA for Bothropstoxin-1 (BthTx-1) expressed the recombinant product as IBs. In order to obtain the native toxin, insoluble and aggregated protein was refolded using high hydrostatic pressure (HHP). IBs were dissolved and refolded (2 kbar, 16 h), and the effects of protein concentration, as well as changes in ratio and concentration of oxido-shuffling reagents, guanidine hydrochloride (GdnHCl), and pH in the refolding buffer, were assayed. A 32% yield (7.6 mg per liter of bacterial culture) in refolding of the native BthTx-1 was obtained using optimal conditions of the refolding buffer (Tris–HCl buffer, pH 7.5, containing 3 mM of a 2:3 ratio of GSH/GSSG, and 1 M GdnHCl). Scanning electron microscopy (SEM) showed that that disaggregation of part of IBs particles occurred upon compression and that the morphology of the remaining IBs, spherical particles, was not substantially altered. Dose-dependent cytotoxic activity of high-pressure refolded BthTx-1 was shown in C2C12 muscle cells.

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Refolding by High Pressure of a Toxin Containing Seven Disulfide Bonds: Bothropstoxin-1 from Bothrops jararacussu

Balduino

Spencer

Malavasi

et al. 2010

Mol Biotechnol

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The effect of the myotoxic Lys49 phospholipase A2 from Agkistrodon contortrix laticinctus snake venom on Na+/K+-ATPase activity of toad bladders

Leite

Pinheiro

Fernandes

et al. 2006

Toxicology in Vitro

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Lys49 myotoxins, secreted phospholipase A2-like proteins of viperid venoms: A comprehensive review

Lomonte

2023

Toxicon

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Effect of a recombinant Lys49PLA2 myotoxin and Lys49PLA2-derived synthetic peptides from Agkistrodon species on membrane permeability to water

Cited by 4 publications

References 9 publications

Refolding by High Pressure of a Toxin Containing Seven Disulfide Bonds: Bothropstoxin-1 from Bothrops jararacussu

Refolding by High Pressure of a Toxin Containing Seven Disulfide Bonds: Bothropstoxin-1 from Bothrops jararacussu

The effect of the myotoxic Lys49 phospholipase A2 from Agkistrodon contortrix laticinctus snake venom on Na+/K+-ATPase activity of toad bladders

Lys49 myotoxins, secreted phospholipase A2-like proteins of viperid venoms: A comprehensive review

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