Expression of the sucrose binding protein from soybean: renaturation and stability of the recombinant protein.
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 beta-strand structure (42%) over the alpha-helix (9.9%). The fluorescence emission 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 7.6M 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.
Rocha
Carolina S
CS
Departamento de Bioquímica e Biologia Molecular/BIOAGRO, Universidade Federal de Viçosa, 36571.000 Viçosa - MG, Brazil.
Luz
Dirce F
DF
Oliveira
Marli L
ML
Baracat-Pereira
Maria C
MC
Medrano
Francisco Javier
FJ
Fontes
Elizabeth P B
EP
D000595:Amino Acid Sequence; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002942:Circular Dichroism; D015870:Gene Expression; D008969:Molecular Sequence Data; D011485:Protein Binding; D011489:Protein Denaturation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D030262:Soybean Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013025:Glycine max / Q000235:genetics / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013395:Sucrose / Q000378:metabolism*
D002352:Carrier Proteins; D011994:Recombinant Proteins; D030262:Soybean Proteins; D013395:Sucrose