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Cloning, expression, and purification of the Staphylococcus simulans lysostaphin using the intein-chitin-binding
P Szweda1, R Pladzyk, R Kotlowski
1Department of Microbiology, Technical University of Gdańsk, ul. Narutowicza 11/12, 80-952 Gdańsk, Poland.
Protein Expression and Purification
|August 3, 2001
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.