Efficient co-expression of a recombinant staphopain A and its inhibitor staphostatin A in Escherichia coli.
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.
Wladyka
Benedykt
B
Department of Analytical Biochemistry, Faculty of Biotechnology, Jagiellonian University, 7 Gronostajowa St., 30-387, Krakow, Poland. patatajj@interia.pl
Puzia
Katarzyna
K
Dubin
Adam
A
D001426:Bacterial Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002846:Chromatography, Affinity; D003546:Cysteine Endopeptidases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D015853:Cysteine Proteinase Inhibitors / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression / Q000235:genetics*; D009154:Mutation / Q000235:genetics; D020673:Protein Renaturation; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013211:Staphylococcus aureus / Q000201:enzymology* / Q000235:genetics
D001426:Bacterial Proteins; D015853:Cysteine Proteinase Inhibitors; D011994:Recombinant Proteins; C501296:staphostatin A, Staphylococcus aureus; D003546:Cysteine Endopeptidases; C501295:staphopain A protein, Staphylococcus aureus