Improved Sequence for Rapid Consistent Purification of Equine Encephalomyelitis Virus Protein. ∗

Abstract

The procedure described here for purification of equine encephalomyelitis virus protein 1 yields consistently a product of high molecular homogeneity. Advantage is taken chiefly of the following principles: (1) prolonged extraction to aid in eliminating the normal chick tissue component; 2 (2) extraction, fractionation and solution of the protein in a balanced salt solution, mammalian Ringer, 3 instead of 0.9% NaCl or buffer salt solutions; (3) filtration of crude extracts with celite to remove mucoid and colloid materials; and (4) aggregation or partial precipitation of the protein in slightly acid medium prior to the first ultracentrifugal cycle.

Diseased embryo tissue ground in the cold in the usual way, 2 is suspended in 4 times its volume of normal Ringer's solution made to pH 9.0 with NH₄OH without buffer salts. After extraction at 5°C for 72 to 96 hours, gross material is eliminated in the angle centrifuge. To each 100 cc of the turbid supernatant fluid, 5 g of No. 512 Celite Filter Aid (Johns-Manville Co., N. Y.) are added and the suspension is filtered with suction through a 1 to 2 mm mat of No. 503 Celite. Standard celite then added to the filtrate, 2 g per 100 cc, is filtered off through a mat also of standard celite. Sometimes the latter step is repeated to obtain an entirely clear filtrate.

The filtrate is acidified to pH 6.5 with 0.2 N HC1, and 120 cc of it distributed immediately in 8 collodion tubes is spun at 17,000 g for 45 minutes. The pellets are taken up in 30 cc of Ringer's solution. In 2 tubes, the solution is spun at 17,000 g for 5 minutes. The supernatant fluid diluted to 60 cc is ultracentrifuged 30 minutes at 67,000 g and the resulting pellets are taken up in 15.0 cc of Ringer fluid.