Purification of a membrane-associated serine esterase from murine cytotoxic T lymphocytes by a single reverse phase columnProtein Expression and Purification
ORCiDDavid M. Ojcius: 0000-0003-1461-4495
AbstractThe plasma and organelle membranes of a cytotoxic T lymphocyte line, CTLL-R8, were isolated by subcellular fractionation. After dissolving in detergent-containing buffer, the membrane proteins were isolated by high-performance liquid chromatography on a single reverse-phase column. The serine esterase activity in the fractions was detected by measuring hydrolysis of the ester compound Nα-benzyloxycarbonyl-l-lysine thiobenzyl ester. A major band was revealed in the fraction with highest serine esterase activity. Under sodium dodecyl sulfate-polyacrylamide gel electrophoresis, this band assumes a molecular weight of about 30 kDa. The amino-terminal sequence of the protein was analyzed and shows 100% identity with that of MCSP-3/granzyme F, a soluble serine esterase previously identified in the cytoplasmic granules of cytotoxic T lymphocytes. Modifications of this reverse-phase column method would thus represent a simple, convenient strategy for obtaining high yields of all the lymphocyte surface proteases, which could then be further characterized for function.
Citation InformationShibo Jiang, Cynthia S. Hasselkus-Light, David M. Ojcius and John Ding-E. Young. "Purification of a membrane-associated serine esterase from murine cytotoxic T lymphocytes by a single reverse phase column" Protein Expression and Purification Vol. 1 Iss. 1 (1990) p. 77 - 80 ISSN: 1046-5928
Available at: http://works.bepress.com/david-ojcius/188/