Trimeric glycoproteins of bean seed storage protein phaseolin were purified from baculovirus-infected insect Sf9 cells for use of structural study

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We demonstrated here insect Sf9 cells provide a useful source of a large quantity of homogeneous plant protein to be used for further structural analysis by X-ray crystallography and other biophysical probes. The seed storage protein phaseolin accumulates in common bean ( Phaseolus vulgaris L.) as a trimeric glycoprotein in the vacuolar protein bodies of developing cotyledon. Here we characterized the post-translational modifications of phaseolin, glycosylation and trimer formation, after expression in Sf9 cells of fall armyworm ( Spodoptera frugiperda) infected by baculovirus. When a cDNA for the mature phaseolin protein (without its own signal peptide) was placed under control of the signal peptide of viral protein GP67 in baculovirus transfer vectors pAcGP67A, phaseolin accumulated within cells at a high level (40 μg/mL). To facilitate the protein purification, six histidines were added to the carboxyl terminal of phaseolin coding sequence as a metal-chelating affinity tag. Phaseolin was extracted from Sf9 cells by 6.0 M guanidinium chloride or 4.0 M urea as a protein solubilizing agent not as a denaturant, and purified by step-wise elution from a nickel column. Phaseolin was modified by a high-mannose glycan at two potential glycosylation-sties in insect Sf9 cells as demonstrated by digestion with endoglycosidase H or peptide N-glycosidase F. Asn 228 and Asn 317 of two potential glycosylation-sites were converted either singly or simultaneously to Glu by site-directed mutagenesis of the cDNA. Similar amounts of wild-type and glycosylation-minus mutants were purified from Sf9 cells. Analytical equilibrium centrifugation analysis demonstrated trimer formation of both wild-type and glycosylation-minus phaseolin. The results indicate that glycosylation is not required for the protein stability or trimer formation of phaseolin. When phaseolin was expressed under control of its own signal peptide in a second transfection vector pAcSG2, phaseolin was accumulated within cells similarly to the first constructs. However, elimination of two but not one glycosilation-sites resulted in the endoproteolytic cleavage(s) of the mature protein. Circular dichroism analysis indicated the proper secondary structure formation of phaseolin in insect Sf9 cells. Taken together, phaseolin was glycosylated, folded into the proper tertiary structure, and assembled into a trimer in insect Sf9 cells.