Isolation of a human serum protein that inhibits the growth of Cryptococcus neoformans

Human serum at 5 to 10% (v/v) in tissue culture medium RPMI-1640, inhibits the growth of Cryptococcus neoformans by 80 to 93%. Serum fractionated on m olecular sieve columns (Sephadex G-200) yielded an active protein fraction. This fraction at 100 mu g protein/ml inhibited the growth of C. neoformans by 54%. When an active G-200 fraction was applied to a dye affinity column (Affi-Gel Blue) the fraction with inhibitory activity was bound by the col umn and was eluted with 1.4 M NaCl in 0.1 M phosphate buffer (pH 7.4). The bound fraction at 62.5 mu g protein/ml inhibited C. neoformans growth by 82 %. On native polyacrylamide gel electrophoresis (Nu-PAGE) the bound fractio n migrated as a major and a minor band. Under the reducing conditions of so dium dodecyl sulfate (SDS)-PAGE the bound fraction yielded 4 prominent band s with MW ranging from 175 kDa to 45 kDa. Purification of the active Sephad ex G-200 peak was achieved using an anion exchange column (DEAE-Sephacel). Protein eluted with 0.1 M NaCl had strong anticryptococcal activity (12.5 m u g/ml, 79% inhibition), which in SDS-PAGE migrated as a single band with a n approximate MW of 85 kDA. This protein appears important in natural host resistance to C. neoformans and polymorphisms or deficiencies may have epid emiologic and diagnostic relevance.