Expressão heteróloga e caracterização bioquímica da proteína recombinante GmSBP2/S64 da soja (Glycine max) / Heterologous expression and biochemical characterization of GmSBP2/S64 recombinant protein from soybean (Glycine max)

AUTOR(ES)
DATA DE PUBLICAÇÃO

2006

RESUMO

The sucrose binding protein (SBP) belongs to the cupin family of proteins and is structurally related to vicilin-like storage proteins. In this investigation, SBP was expressed in E. coli and large amounts of the protein accumulated in the insoluble fraction as aggregated, denatured protein. The refolding of the purified protein proceeded with a progressive removal of urea into the renaturation buffer, which contained an oxido shuffling system (2 mM reduced glutathione and 0.2 mM oxidized glutathione) and glycerol 5% (v/v) as a proteinstabilizing agent. The renaturation of the protein was assayed by using far-UV circular dichroism (CD), intrinsic fluorescence, and quenching by acrylamide, KCl and KI. The percentage of secondary structure of the renatured protein, which was calculated on the basis of the CD spectrum, was consistent with that obtained by theoretical modeling with a large predominance of beta-strand structure (42%) over the alfa-helix (9.9%). The fluorescence emission maximum of 303 nm for SBP indicated that the fluorescent tryptophan was completely buried within a highly hydrophobic environment. Nevertheless, tryptophan quenching by acrylamide, KI and CsCL and the respective Stem-Volmer (Ksv) constant of 23.5 +- 0.5 M-1, 16.1 +- 0.2 M-1 and 4.9 +- 0.1 M-1 indicate that the fluorescent tryptophan residues were quite accessible to the quenchers and hence exposed to the solvent. We also measured the equilibrium dissociation constant (Kd) of sucrose binding by fluorescence titration using the refolded protein. The low sucrose binding affinity (Kd = 2.79 +- 0.22 mM) of the renatured protein was similar to that of the native protein purified from soybean seeds. Collectively, our results indicate that the folded structure of the renatured protein is similar to the native SBP protein. As a member of the bicupin family of proteins which include highly stable seed storage proteins, it was of interest to determine the structural stability of SBP. The thermal and chemical denaturations of the protein were examined by monitoring changes in the CD spectra and in the intrinsic fluorescence of the renatured protein. Our results indicate that SBP remained folded to a similar extent in the presence or absence of 8 M urea or 6 M GdnHCl. Likewise, it was fairly stable to high temperatures. The high stability of the renatured protein may be a eminiscent property of SBP from its evolutionary relatedness to the seed storage proteins.

ASSUNTO(S)

dicroísmo circular circular dichroism fluorescência proteína recombinante recombinant protein fluorescence biologia molecular

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