title: Refolding by high pressure of a toxin containing seven disulfide bonds: bothropstoxin-1 from Bothrops jararacussu.
abstract: 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.
authors.affiliation: Centro de Biotecnologia, Instituto de Pesquisas Energéticas e Nucleares-CNEN/SP, Av. Professor Lineu Prestes 2242, São Paulo, SP 05508-000, Brazil.
authors.forename: Keli N
authors.initials: KN
authors.lastname: Balduino
authors.forename: Patrick J
authors.initials: PJ
authors.lastname: Spencer
authors.forename: Natalia V
authors.initials: NV
authors.lastname: Malavasi
authors.forename: Rosa M
authors.initials: RM
authors.lastname: Chura-Chambi
authors.forename: Laura S
authors.initials: LS
authors.lastname: Lemke
authors.forename: Ligia
authors.initials: L
authors.lastname: Morganti
mesh_terms: D000818:Animals; D017837:Bothrops / Q000378:metabolism*; D016923:Cell Death / Q000187:drug effects; D002460:Cell Line; D003435:Crotalid Venoms / Q000737:chemistry* / Q000378:metabolism / Q000494:pharmacology; D004220:Disulfides / Q000737:chemistry / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D005978:Glutathione; D019791:Guanidine; D006863:Hydrogen-Ion Concentration; D006874:Hydrostatic Pressure; D002479:Inclusion Bodies / Q000737:chemistry; D051379:Mice; D008855:Microscopy, Electron, Scanning; D058849:Protein Refolding; D055550:Protein Stability; D011994:Recombinant Proteins / Q000737:chemistry* / Q000378:metabolism / Q000494:pharmacology
chemical_list: D003435:Crotalid Venoms; D004220:Disulfides; D011994:Recombinant Proteins; C078097:bothropstoxin; D005978:Glutathione; D019791:Guanidine