Abstract
Cysteine proteases are implicated in many regulatory and degradative processes in animal and plant cells. Many of the proteases are strongly inhibited by an irreversible inhibitor, trans-(epoxysuccinyl)-L-leucylamino-4-guanidinobutane (E-64) from Aspergillus japonicus. Here we report a method for purification of cysteine proteases by affinity chromatography on E-64. Attachment of the inhibitor to thiopropyl Sepharose through its epoxy group resulted in the loss of its irreversible activity but did not affect the specificity of interaction or its capability to bind cysteine proteases. Papain that served as a model cysteine protease was fully active after elution. We also provide evidence for purification of active proteases from a mixture of extracellular fluid of Botrytis cinerea- and Trichoderma harzianum-inoculated bean plants. Since the proteases are eluted with urea after the column is washed with 1 M NaCl, this procedure may provide highly efficient purification.
| Original language | English |
|---|---|
| Pages (from-to) | 247-250 |
| Number of pages | 4 |
| Journal | Protein Expression and Purification |
| Volume | 15 |
| Issue number | 3 |
| DOIs | |
| State | Published - Apr 1999 |
Bibliographical note
Funding Information:We thank Dr. Gilon (Department of Chemistry, Hebrew University) for the advice on the chemistry of the reactions, Dr. Kaplan (Department of Plant Sciences) for critical discussion of the method, and Dr. Elad (Volcani Institute, Israel) for the samples of bean plants inoculated with B. cinerea and T. harzianum. This work was supported in part by a grant from the Binational US–Israel Science foundation (BSF).