Purification of the stress protein alpha B-crystallin and its differentially phosphorylated forms.
The stress protein alpha B-crystallin was recently identified as a component of central nervous system myelin that is strongly immunogenic to human T cells. Stress-induced alpha B-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 alpha B-crystallin. For immunological studies, therefore, the availability of purified preparations of alpha B-crystallin and its various differentially phosphorylated forms would be especially useful. Here we describe a rapid and simple method for the purification of alpha B-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 alpha B-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 alpha B-crystallin into purified fractions with a defined number of phosphorylated serines.
van Noort
J M
JM
Division of Immunological and Infectious Diseases, TNO Prevention and Health, Leiden, The Netherlands. jm.vannoort@pg.tno.nl
van Veelen
P
P
Hopstaken
F
F
D000818:Animals; D002417:Cattle; D002850:Chromatography, Gel / Q000379:methods; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D002852:Chromatography, Ion Exchange / Q000379:methods; D003459:Crystallins / Q000302:isolation & purification* / Q000378:metabolism; D007908:Lens, Crystalline / Q000737:chemistry*; D013058:Mass Spectrometry; D010766:Phosphorylation
D003459:Crystallins