Use este identificador para citar ou linkar para este item:
Tipo: Artigo
Título: Expression of the sucrose binding protein from soybean: renaturation and stability of the recombinant protein
Autor(es): Rocha, Carolina S.
Luz, Dirce F.
Oliveira, Marli L.
Baracat-Pereira, Maria C.
Medrano, Francisco Javier
Fontes, Elizabeth P.B.
Abstract: The sucrose binding protein (SBP) belongs to the cupin family of proteins and is structurally related to vicilin-like storage proteins. In this investigation, a SBP isoform (GmSBP2/S64) was expressed in E. coli and large amounts of the protein accumulated in the insoluble fraction as inclusion bodies. The renatured protein was studied by circular dichroism (CD), intrinsic fluorescence, and binding of the hydrophobic probes ANS and Bis-ANS. The estimated content of secondary structure of the renatured protein was consistent with that obtained by theoretical modeling with a large predominance of b-strand structure (42%) over the a-helix (9.9%). The fluorescence emis- sion maximum of 303 nm for SBP2 indicated that the fluorescent tryptophan was completely buried within a highly hydrophobic environment. We also measured the equilibrium dissociation constant (K d ) of sucrose binding by fluorescence titration using the refolded protein. The low sucrose binding affinity (K d = 2.79 ± 0.22 mM) of the renatured protein was similar to that of the native protein purified from soybean seeds. Collectively, these results indicate that the folded structure of the renatured protein was similar to the native SBP protein. As a member of the bicupin family of proteins, which includes highly stable seed storage proteins, SBP2 was fairly stable at high temperatures. Likewise, it remained folded to a similar extent in the presence or absence of 7.6 M urea or 6.7 M GdmHCl. The high stability of the renatured protein may be a reminiscent property of SBP from its evolutionary relatedness to the seed storage proteins.
Palavras-chave: Glycine max
Heterologous expression
Protein renaturation
Cupin structure
Sucrose binding protein
Editor: Phytochemistry
Tipo de Acesso: Elsevier Ltd
Data do documento: 12-Jan-2007
Aparece nas coleções:Artigos

Arquivos associados a este item:
Arquivo Descrição TamanhoFormato 
  Until 2100-12-31
texto completo1,05 MBAdobe PDFVisualizar/Abrir    Solictar uma cópia

Os itens no repositório estão protegidos por copyright, com todos os direitos reservados, salvo quando é indicado o contrário.