Biophysical characterization and formulation of F1-V, a recombinant plague antigen.
The recombinant plague antigen, F1-V, was studied for its structural characteristics using several biophysical techniques. A larger apparent molecular weight relative to its calculated molecular weight obtained from size exclusion chromatography, an unusually large R(g) obtained from MALS, and ANS dye binding studies which indicate that all hydrophobic regions of the protein are exposed to solvent demonstrated that F1-V exists like a disordered protein with a worm-like conformation. The pH-solubility profile of F1-V showed a solubility minimum at pH 5, close to its pI, consistent with the lack of repulsive forces that result in aggregation. Thus, in contrast to most globular proteins that exhibit a secondary and a tertiary structure, F1-V seems to lack tertiary structure and like an unfolded protein is more prone to aggregation via hydrophobic interactions. Despite this, when renatured gradually using descending guanidine hydrochloride concentration dialysis, in the presence of Mg+2, a surfactant and arginine hydrochloride at a pH of 7.5, F1-V appears to populate predominantly in its monomeric state.
D'Souza
Ajit Joseph M
AJ
BD Technologies, 21 Davis Dr., Research Triangle Park, North Carolina 27519, USA. ajit_dsouza@bd.com
Ford
Brandi M
BM
Mar
Kevin D
KD
Sullivan
Vince J
VJ
D000595:Amino Acid Sequence; D000942:Antigens, Bacterial / Q000737:chemistry* / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics; D002850:Chromatography, Gel; D008969:Molecular Sequence Data; D010930:Plague / Q000235:genetics / Q000517:prevention & control; D010931:Plague Vaccine / Q000737:chemistry* / Q000235:genetics; D011489:Protein Denaturation; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry* / Q000235:genetics; D015010:Yersinia pestis / Q000737:chemistry / Q000235:genetics / Q000472:pathogenicity
D000942:Antigens, Bacterial; D001426:Bacterial Proteins; D010931:Plague Vaccine; D011994:Recombinant Proteins