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

The stress protein alpha B-crystallin was recently identified as a componen t of central nervous system myelin that is strongly immunogenic to human T cells. Stress-induced alpha B-crystallin that accumulates in the central ne rvous system is phosphorylated and recent evidence indicates that both rode nt and human T cells can discriminate between differentially phosphorylated forms of alpha B-crystallin. For immunological studies, therefore, the ava ilability of purified preparations of alpha B-crystallin and its various di fferentially phosphorylated forms would be especially useful. Here we descr ibe a rapid and simple method for the purification of alpha B-crystallin fr om adult bovine eye lenses by a combination of size-exclusion chromatograph y and reversed-phase high-performance liquid chromatography. This yields a preparation of purified alpha B-crystallin that contains all the various di fferentially phosphorylated forms of the protein. Subsequent anion-exchange chromatography under denaturing conditions permits the separation of these phosphorylated forms of alpha B-crystallin into purified fractions with a defined number of phosphorylated serines. (C) 1998 Elsevier Science B.V. Al l rights reserved.