Your browser doesn't support javascript.
loading
Effect of disulfide bonds on the structure, function, and stability of the trypsin/tPA inhibitor from Erythrina caffra: site-directed mutagenesis, expression, and physiochemical characterization.
Lehle, K; Kohnert, U; Stern, A; Popp, F; Jaenicke, R.
Afiliação
  • Lehle K; Institut für Biophysik und Physikalische Biochemie, Universität Regensburg, Germany.
Nat Biotechnol ; 14(4): 476-80, 1996 Apr.
Article em En | MEDLINE | ID: mdl-9630923
ABSTRACT
Erythrina trypsin/tPA inhibitor (ETI) from the seeds of Erythrina caffra retains its native structure and inhibitory function after reducing its two disulfide bonds. In order to elucidate the specific role of these crosslinks, alanine residues were substituted for cysteines after cloning the gene in Escherichia coli. Expression of the recombinant inhibitor and the substitution mutants, C83A, CC39, 83AA, and CC132, 139AA, led to inclusion bodies. After solubilization in guanidinium-chloride (GdmCl)/dithiothreitol and oxidation in glutathione buffer, activity could be recovered at yields up to 80%. The mutant proteins exhibit full inhibitory function without detectable alterations of their native structure. However, their stability is reduced at acid pH, where the oxidized natural inhibitor retains its native structure, the reduced wildtype protein and the mutants undergo at least partial denaturation, reflected by decreased pH ranges of stability pH 5-7 for the reduced inhibitor, pH 2.5-8.5 for CC132, 139AA, and pH 3.5-8.5 for C83A and CC39, 83AA. Urea and GdmCl denaturation at pH 7 show hysteresis for both the oxidized inhibitor and the double mutant CC132, 139AA. In contrast, the reduced protein and the other mutants exhibit true equilibrium transitions at pH 7, with urea half-concentrations of 0.9 M and 1.9 M and GdmCl half-concentrations of 0.5 M and 1.0 M, respectively. The stability of Erythrina trypsin/tPA inhibitor follows the sequence oxidized ETI > CC132, 139AA > CC39, 83AA and C83A > reduced ETI.
Assuntos
Buscar no Google
Coleções: 01-internacional Base de dados: MEDLINE Assunto principal: Proteínas de Plantas / Plantas Medicinais / Inibidores da Tripsina / Erythrina Idioma: En Revista: Nat Biotechnol Assunto da revista: BIOTECNOLOGIA Ano de publicação: 1996 Tipo de documento: Article País de afiliação: Alemanha
Buscar no Google
Coleções: 01-internacional Base de dados: MEDLINE Assunto principal: Proteínas de Plantas / Plantas Medicinais / Inibidores da Tripsina / Erythrina Idioma: En Revista: Nat Biotechnol Assunto da revista: BIOTECNOLOGIA Ano de publicação: 1996 Tipo de documento: Article País de afiliação: Alemanha