AI Article Synopsis
- The carboxypeptidase T precursor from Thermoactinomyces vulgaris was expressed in E. coli, resulting in about 60 mg of insoluble protein from 1 L of culture.
- This protein was denatured with guanidine-HCl and successfully renatured, with calcium playing a crucial role in stabilizing the refolded enzyme.
- After purification through gel-filtration and processing with subtilisin, the mature carboxypeptidase T produced showed properties similar to the natural enzyme, including its sequence and stability.
Article 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 1 L of culture liquid was about 60 mg. The obtained inclusion bodies were denatured in 6 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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| http://dx.doi.org/10.1016/j.pep.2004.10.020 | DOI Listing |
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