The pro-sequence domain of streptopain directs the folding of the mature enzyme |
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Authors: | Anderson Elizabeth T Winter Laurie A Fernsten Phil Olmsted Stephen B Matsuka Yury V |
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Institution: | Department of Protein Chemistry, Wyeth Research, 401 N. Middletown Road, Bldg. 205/228, Pearl River, NY 10965, USA. |
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Abstract: | The cysteine endopeptidase streptopain, an extracellular enzyme from pathogenic Streptococcus pyogenes, is synthesized as a precursor containing an NH2-terminal pro-sequence. The pro-sequence of streptopain was expressed in Escherichia coli and subjected to structural and functional investigation. Heat-induced denaturation of the pro-sequence studied using circular dichroism spectroscopy revealed that it forms a compact structure and represents an independently folded domain. The isolated pro-sequence exhibits high affinity towards mature streptopain and associates with its cognate enzyme by forming an equimolar complex. Refolding of denatured streptopain in the presence of pro-sequence in vitro facilitated recovery of active enzyme. Expression of the mature streptopain in E. coli either alone, or in trans with its pro-sequence as an independent polypeptide, led to the formation of insoluble protein aggregates or functionally active enzyme, respectively. These results demonstrate that the pro-sequence domain acts as an intramolecular chaperone that directs the correct folding of the mature streptopain. |
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Keywords: | Streptococcal cysteine protease Independently folded domain Refolding Intramolecular chaperone Co-expression |
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