Title

Purification of a membrane-associated serine esterase from murine cytotoxic T lymphocytes by a single reverse phase column

ORCiD

David M. Ojcius: 0000-0003-1461-4495

Document Type

Article

Publication Title

Protein Expression and Purification

ISSN

1046-5928

Volume

1

Issue

1

DOI

10.1016/1046-5928(90)90049-5

First Page

77

Last Page

80

Publication Date

9-1-1990

Abstract

The 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.