Staphopain A is a staphylococcal cysteine protease. Genes encoding staphopain A and its specific inhibitor, staphostatin A, are localized in an operon. Staphopain A is an important staphylococcal virulence factor. It is difficult to perform studies on its interaction with other proteins due to problems in obtaining a sufficient amount of the enzyme from natural sources. Therefore efforts were made to produce a recombinant staphopain A. Sequences encoding the mature form of staphopain A and staphostatin A were PCR-amplified from Staphylococcus aureus genomic DNA and cloned into different compatible expression vectors. Production of staphopain A was observed only when the enzyme was co-expressed together with its specific inhibitor, staphostatin A. Loss of the function mutations introduced within the active site of staphopain A causes the expression of the inactive enzyme. Mutations within the reactive centre of staphostatin A result in abrogation of production of both the co-expressed proteins. These results support the thesis that the toxicity of recombinant staphopain A to the host is due to its proteolytic activity. The coexpressed proteins are located in the insoluble fraction. Ni2+-nitrilotriacetate immobilized metal-affinity chromatography allows for an efficient and easy purification of staphopain A. Our optimized refolding parameters allow restoration of the native conformation of the enzyme, with yields over 10-fold higher when compared with isolation from natural sources.
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Research Article|
December 14 2004
Efficient co-expression of a recombinant staphopain A and its inhibitor staphostatin A in Escherichia coli
Benedykt WLADYKA;
Benedykt WLADYKA
1
1Department of Analytical Biochemistry, Faculty of Biotechnology, Jagiellonian University, 7 Gronostajowa St., 30-387, Krakow, Poland
1To whom correspondence should be addressed (email patatajj@interia.pl).
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Katarzyna PUZIA;
Katarzyna PUZIA
1Department of Analytical Biochemistry, Faculty of Biotechnology, Jagiellonian University, 7 Gronostajowa St., 30-387, Krakow, Poland
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Adam DUBIN
Adam DUBIN
1Department of Analytical Biochemistry, Faculty of Biotechnology, Jagiellonian University, 7 Gronostajowa St., 30-387, Krakow, Poland
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Publisher: Portland Press Ltd
Received:
June 07 2004
Revision Received:
August 17 2004
Accepted:
August 23 2004
Accepted Manuscript online:
August 23 2004
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London
2005
Biochem J (2005) 385 (1): 181–187.
Article history
Received:
June 07 2004
Revision Received:
August 17 2004
Accepted:
August 23 2004
Accepted Manuscript online:
August 23 2004
Citation
Benedykt WLADYKA, Katarzyna PUZIA, Adam DUBIN; Efficient co-expression of a recombinant staphopain A and its inhibitor staphostatin A in Escherichia coli. Biochem J 1 January 2005; 385 (1): 181–187. doi: https://doi.org/10.1042/BJ20040958
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