An improved purification procedure for cyclosporine synthetase

Velkov, Tony, Singaretnam, Lloyd George and Lawen, Alfons 2006, An improved purification procedure for cyclosporine synthetase, Protein expression and purification, vol. 45, no. 2, pp. 275-287, doi: 10.1016/j.pep.2005.07.012.

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Title An improved purification procedure for cyclosporine synthetase
Author(s) Velkov, Tony
Singaretnam, Lloyd George
Lawen, Alfons
Journal name Protein expression and purification
Volume number 45
Issue number 2
Start page 275
End page 287
Publisher Academic Press
Place of publication San Diego, Calif.
Publication date 2006-02
ISSN 1046-5928
Keyword(s) cyclosporin synthetase
non-ribosomal peptide synthetase
antibiotic biosynthesis
in vitro enzymatic biosynthesis
large-scale protein purification
fungal biotechnology
tolypocladium inflatum
Summary We have developed expedient and reliable methods to isolate cyclosporin synthetase for in vitro biosynthesis of cyclosporins. We have examined enzyme purification strategies suited to large-scale processing and present a chromatographic sequence that serves as a pilot model for industrial scale preparation of cyclosporin synthetase from cyclosporin producing fungi. A chromatographic sequence consisting of ammonium sulfate precipitation → gel filtration → hydrophobic interaction chromatography → anion exchange chromatography, yielded an electrophoretically homogeneous cyclosporin synthetase preparation (Coomassie G-250 brilliant blue staining). Furthermore, a native polyacrylamide gel electrophoresis system was developed for the isolation of active cyclosporin synthetase enzyme from crude extracts of cyclosporin producing fungi. The environmental factors affecting enzyme stability and the continuity of the in vitro cyclosporin biosynthetic reaction-temperature, pH, and substrate depletion were assessed and manageable conditions have been defined for sustainable cyclosporin biosynthesis with enzyme isolates. Cyclosporin synthetase exhibited an optimal temperature range of 24–29 °C and a pH optimum of 7.6. The native enzyme displayed a pI of 5.7, as determined by isoelectric focusing. The industrial implementation of an in vitro biosynthetic approach could potentially prove useful for the production of important therapeutic cyclosporins which occur as only minor fermentation by-products.
Language eng
DOI 10.1016/j.pep.2005.07.012
Field of Research 060107 Enzymes
060199 Biochemistry and Cell Biology not elsewhere classified
060702 Plant Cell and Molecular Biology
HERDC Research category C1.1 Refereed article in a scholarly journal
Copyright notice ©2005, Elsevier
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Document type: Journal Article
Collections: Faculty of Health
School of Medicine
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