Article ID | Journal | Published Year | Pages | File Type |
---|---|---|---|---|
10843509 | Protein Expression and Purification | 2005 | 9 Pages |
Abstract
Carboxypeptidase T precursor from Thermoactinomyces vulgaris, which fails to contain its own leader peptide, has been expressed in Escherichia coli as insoluble cytoplasmic inclusion bodies. The yield of a washed recombinant protein from 1Â L of culture liquid was about 60Â mg. The obtained inclusion bodies were denatured in 6Â M guanidine-HCl and then renatured by a rapid dilution. The important role of calcium for the complete stabilization of the refolded carboxypeptidase T precursor was established. After removal of minor admixture proteins by gel-filtration through Superdex 75, an electrophoretically homogeneous preparation of the native precursor of carboxypeptidase T was obtained. Processing of the resulting protein by subtilisin led to the formation of the mature carboxypeptidase T in which N-terminal sequence, molecular size, thermal stability, and catalytic properties were comparable to those of the natural enzyme.
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Authors
Lesya Trachuk, Andrei Letarov, Irina A. Kudelina, Margarita P. Yusupova, Galina G. Chestukhina,