| Autor: |
Rank, Kenneth B., Mildner, Ana M., Leone, Joseph W., Koeplinger, Kenneth A., Chou, K.C., Tomasselli, Alfredo G., Heinrikson, Robert L., Sharma, Satish K. |
| Zdroj: |
Protein Expression and Purification; July 2001, Vol. 22 Issue: 2 p258-266, 9p |
| Abstrakt: |
We report here the cloning and high-level expression of a soluble proform of human caspase 3 (Ser24-H277) engineered to contain a short stretch of N-terminal sequence (MTISDSPREQD) from the prosegment of procaspase 8 and a C-terminal heptahistidine tag. The precursor protein isolated from extracts of recombinant Escherichia coliby immobilized metal-ion affinity chromatography was predominantly unprocessed and migrated as a 32-kDa polypeptide on sodium dodecyl sulfate-polyacrylamide gels. Incubation of this protein with recombinant human caspase 8 produced fragments characteristic of the properly processed caspase 3, but the product was inactive. Amino-terminal sequence analysis of the caspase 3 polypeptides proved that caspase 8 had specifically cleaved the Asp175-Ser176bond to yield the expected p18 and p12 subunits, with partial cleavage at the Asp28-Ser29bond to release the prosegment. The lack of caspase 3 activity was found to be the result of a fortuitous mutation in which Trp206in the S4 subsite was replaced by arginine (W206R). This mutant procaspase 3, which we call m-pro3, serves as a useful reagent with which to test the efficacy of caspase 8 inhibitors in blocking processing of the natural polypeptide substrate of this enzyme and may be valuable as a source of “proenzyme” for crystallographic analysis. |
| Databáze: |
Supplemental Index |
| Externí odkaz: |
|
Full Text from ScienceDirect