PMID: 8463263Apr 5, 1993Paper

Isolation and characterization of recombinant human cathepsin E expressed in Chinese hamster ovary cells.

The Journal of Biological Chemistry
T TsukubaK Yamamoto

Abstract

The cDNA sequence encoding precursor forms of human cathepsin E (CE), an intracellular aspartic proteinase, was expressed in Chinese hamster ovary cells using an SV40 promotor-driven expression vector. By immunoelectron microscopic studies using an anti-human CE antibody and by Percoll density gradient fractionation, the expressed CE was found to be in two different intracellular fractions; the cytosolic compartment and the vacuolar system. The CEs in both the cytosolic and the vacuolar fractions were highly purified by a simple method involving Percoll density gradient fractionation, chromatography on concanavalin A-Sepharose, Mono Q, and TSK-GelG2000SW, and termed s-CE and v-CE, respectively. The v-CE was further separated into a major (v-CE1) and a minor (v-CE2) form by Mono Q chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting revealed that the s-CE and v-CE1 consists of two polypeptides of 90 and 84 kDa, whereas v-CE2 is composed of 84- and 82-kDa polypeptides. The NH2-terminal amino acid sequence analyses showed that the 90- and 84-kDa proteins from both s-CE and v-CE started with Ser3 and Lys30 of the sequence of human gastric CE predicted from its cDNA sequence, respectively, and...Continue Reading

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