Human carbonic anhydrase IV: in vitro activation and purification of disulfide-bonded enzyme following expression in Escherichia coli.
Human carbonic anhydrase IV (CA IV) expressed in Escherichia coli was refolded and activated in cell extracts with the help of endogenous periplasmic protein disulfide isomerase, DsbA, in the presence of oxidized glutathione. The refolding and activation were inhibited by bacitracin but not affected by known cofactors or activators of other chaperones. Although the yield of the purified CA IV recovered from cell extracts was maximal when activated at 4 degrees C in the presence of 2 mM oxidized glutathione, the rate of refolding and activation was much more rapid at 25 and 37 degrees C. The enzyme purified from the E. coli cell extracts following activation in vitro showed similar structural stability and functional properties as CA IV purified from secretion medium from a stably transfected CHO cell line. These studies suggest that the soluble truncated form of human CA IV expressed in E. coli, which is disulfide-bonded zinc metalloenzyme, can provide a useful model enzyme for studies of protein folding and enzyme activation in vitro. Furthermore, the procedure described for recovery of CA IV following expression in E. coli may be useful for in vitro activation and subsequent purification of other disulfide-containing proteins.
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Edward A. Doisy Department of Biochemistry and Molecular Biology, St. Louis University, School of Medicine, Missouri 63104, USA.
Pham
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Okuyama
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Sly
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D001414:Bacitracin / Q000494:pharmacology; D002256:Carbonic Anhydrases / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification*; D003001:Cloning, Molecular; D004220:Disulfides / Q000737:chemistry*; D004789:Enzyme Activation / Q000187:drug effects; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D005978:Glutathione / Q000031:analogs & derivatives / Q000378:metabolism / Q000494:pharmacology; D019803:Glutathione Disulfide; D017261:Glycosylphosphatidylinositols / Q000378:metabolism; D006801:Humans; D007535:Isomerases / Q000187:drug effects / Q000378:metabolism; D018832:Molecular Chaperones / Q000378:metabolism; D019704:Protein Disulfide-Isomerases; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D013696:Temperature
D004220:Disulfides; D017261:Glycosylphosphatidylinositols; D018832:Molecular Chaperones; D011994:Recombinant Proteins; D001414:Bacitracin; D002256:Carbonic Anhydrases; D007535:Isomerases; D019704:Protein Disulfide-Isomerases; D005978:Glutathione; D019803:Glutathione Disulfide