Combined use of trypsin-agarose affinity chromatography and reversed-phase high-performance liquid chromatography for the purification of single-chain protease inhibitor from corn seeds.
We have developed a large-scale method for recovering the corn inhibitor of trypsin and activated Hageman factor from a trypsin-agarose column predominantly in the single-chain form. To do so, inhibitor retained by the column was eluted with 1.0 M glycine buffer, pH 2.1. We have used reversed-phase high-performance liquid chromatography to further purify the inhibitor eluted from the trypsin-agarose column by separating the single-chain inhibitor from two-chain inhibitor (a small amount of which is present in the preparation after trypsin-agarose chromatography) and from still smaller amounts of another protein (apparently trypsin) that appears as a contaminant during trypsin-agarose chromatography.
D000596:Amino Acids / Q000032:analysis; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D004800:Enzymes, Immobilized; D011480:Protease Inhibitors / Q000302:isolation & purification*; D012685:Sepharose; D014357:Trypsin; D003313:Zea mays / Q000032:analysis*
D000596:Amino Acids; D004800:Enzymes, Immobilized; D011480:Protease Inhibitors; D012685:Sepharose; D014357:Trypsin