Purification of the stress protein αB-crystallin and its differentially phosphorylated forms

Abstract The stress protein αB-crystallin was recently identified as a component of central nervous system myelin that is strongly immunogenic to human T cells. Stress-induced αB-crystallin that accumulates in the central nervous system is phosphorylated and recent evidence indicates that both rodent and human T cells can discriminate between differentially phosphorylated forms of αB-crystallin. For immunological studies, therefore, the availability of purified preparations of αB-crystallin and its various differentially phosphorylated forms would be especially useful. Here we describe a rapid and simple method for the purification of αB-crystallin from adult bovine eye lenses by a combination of size-exclusion chromatography and reversed-phase high-performance liquid chromatography. This yields a preparation of purified αB-crystallin that contains all the various differentially phosphorylated forms of the protein. Subsequent anion-exchange chromatography under denaturing conditions permits the separation of these phosphorylated forms of αB-crystallin into purified fractions with a defined number of phosphorylated serines.