Purification, structure, and properties of hybrid beta-galactosidase proteins.
Structural studies are reported on seven hybrid proteins produced by gene fusions that contain a "foreign" amino acid sequence substituting for part of the NH2-terminal region of the beta-galactosidase polypeptide. All of these hybrid proteins retain beta-galactosidase enzyme activity. A simple and rapid purification scheme for the hybrid beta-galactosidase is described, involving ammonium sulfate fractionation, DEAE-Bio-Gel, and Bio-Gel A-1.5 chromatography. The proteins are tetramers and have activity equivalent to that of wild type enzyme. Their amino acid sequences were determined by isolation and sequence determination of the cyanogen bromide peptide containing the joining site. The subunit sizes vary from 1009 to 1355 residues compared to 1023 for wild type. Up to 26 amino acid residues at the NH2 terminus of beta-galactosidase can be substituted by the new sequence. The nature of the new sequence apparently has no influence on stability or activity of the hybrid, but those hybrids with more of the beta-galactosidase sequence deleted are less stable to heat or urea treatment and tend to dissociate to dimeric form. All hybrids are less stable to heat and urea than wild type. Antipeptide antibodies raised against peptides derived from the NH2-terminal region of wild type beta-galactosidase were found to bind to the hybrid proteins, although they do not bind to the normal enzyme. These results indicate that the quaternary structure is disturbed but not disrupted by substitution of the different sequence, and these results help to localize one of the intersubunit contact regions in beta-galactosidase.
D000595:Amino Acid Sequence; D055598:Chemical Phenomena; D002621:Chemistry; D003488:Cyanogen Bromide; D005696:Galactosidases / Q000302:isolation & purification*; D010446:Peptide Fragments / Q000032:analysis; D001616:beta-Galactosidase / Q000032:analysis / Q000302:isolation & purification*
D010446:Peptide Fragments; D005696:Galactosidases; D001616:beta-Galactosidase; D003488:Cyanogen Bromide