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Refolding and purification of Cephalosporium acremonium deacetoxycephalosporin C synthetase/hydroxylase from granules of recombinant Escherichia coli
Author(s) -
SK Ghag,
DN Brems,
TC Hassell,
WK Yeh
Publication year - 1996
Publication title -
biotechnology and applied biochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.468
H-Index - 70
eISSN - 1470-8744
pISSN - 0885-4513
DOI - 10.1111/j.1470-8744.1996.tb00393.x
Subject(s) - enzyme , biochemistry , escherichia coli , chemistry , recombinant dna , urea , size exclusion chromatography , chromatography , gene
The gene for bifunctional deacetoxycephalosporin C synthetase/hydroxylase of Cephalosporium acremonium was cloned and overexpressed as an insoluble and inactive enzyme in granules of recombinant Escherichia coli. About 40‐60% of expected synthetase activity along with 50‐80% protein purity could be recovered directly from granular extracts with only a single empirically optimized refolding step. Further purification to homogeneity was achieved by a single anion‐exchange‐chromatographic step in the presence of denaturing concentrations of urea. The main obstacle to converting the homogeneous unfolded protein into the active enzyme was a urea‐dependent aggregation during refolding that led to irreversible enzyme inactivation. Information obtained from refolding studies using gel‐filtration HPLC, fluorescence spectroscopy and disulphide analysis led to an optimal enzyme refolding scheme that resulted in a highly active (i.e. 65‐75% of the expected activity) and moderately stable fungal synthetase/hydroxylase.