Cloning, Expression, and Purification of the Staphylococcus simulans Lysostaphin Using the Intein-Chitin-Binding Domain (CBD) System |
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Authors: | Piotr Szweda, Radoslaw Pladzyk, Roman Kotlowski,J zef Kur |
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Affiliation: | b Department of Microbiology;a Department of Food Chemistry and Technology, Technical University of Gdask, ul. Narutowicza 11/12, 80-952, Gdask, Poland |
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Abstract: | The Staphylococcus simulans gene encoding lysostaphin has been PCR amplified from pRG5 recombinant plasmid (ATCC 67076) and cloned into Escherichia coli expression pTYB12 vector (IMPACT-CN System, New England BioLabs) which allows the overexpression of a target protein as a fusion to a self-cleavable affinity tag. The self-cleavage activity of the intein allows the release of the lysostaphin enzyme from the chitin-bound intein tag, resulting in a single-column purification of the target protein. This abundant overproduction allows purifying milligram amounts of the enzyme. |
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Keywords: | purification expression peptidase peptidoglycan staphylococci. |
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