Purification and characterization of hexahistidine-tagged cyclohexanone monooxygenase expressed in Saccharomyces cerevisiae and Escherichia coli

Cheesman, Matthew J., Kneller, M. Byron, Kelly, Edward J., Thompson, Stella J., Yeung, Catherine K., Eaton, David L. and Rettie, Allan E. (2001) Purification and characterization of hexahistidine-tagged cyclohexanone monooxygenase expressed in Saccharomyces cerevisiae and Escherichia coli. Protein Expression and Purification, 21 1: 81-86. doi:10.1006/prep.2000.1340


Author Cheesman, Matthew J.
Kneller, M. Byron
Kelly, Edward J.
Thompson, Stella J.
Yeung, Catherine K.
Eaton, David L.
Rettie, Allan E.
Title Purification and characterization of hexahistidine-tagged cyclohexanone monooxygenase expressed in Saccharomyces cerevisiae and Escherichia coli
Formatted title
Purification and characterization of hexahistidine-tagged cyclohexanone monooxygenase expressed in Saccharomyces cerevisiae and Escherichia coli
Journal name Protein Expression and Purification   Check publisher's open access policy
ISSN 1046-5928
1096-0279
Publication date 2001-02
Sub-type Article (original research)
DOI 10.1006/prep.2000.1340
Volume 21
Issue 1
Start page 81
End page 86
Total pages 6
Place of publication Maryland Heights, MO, United States
Publisher Academic Press
Language eng
Formatted abstract
Cyclohexanone monooxygenase (CMO) is a soluble flavoenzyme originally isolated from Acinetobacter spp. which carries out Baeyer-Villiger reactions with cyclic ketone substrates. In the present study we cloned the Acinetobacter CMO gene and modified it for facile purification from heterologous expression systems by incorporation of a His6-tag at its C-terminus. A single purification step employing metal (Ni2+)-affinity column chromatography provided essentially homogeneous enzyme in yields of 69–72%. The properties of the purified, recombinant enzymes (rCMO) were compared with that of native CMO (nCMO) isolated from Acinetobacter cultures grown in the presence of cyclohexanone. The specific activities of His6-tagged rCMO and nCMO toward their index substrate, cyclohexanone, were similar and ranged from 14 to 20 μmol/min/mg. nCMO and rCMO from the Escherichia coli expression system exhibited molecular masses, determined by electrospray mass spectrometry, of 60,800 and 61,615 Da, respectively, an increase for the recombinant enzyme equivalent to the mass of the His6-tag. However, rCMO expressed in Saccharomyces cerevisiae consistently exhibited a mass some 50 Da larger than rCMO expressed in bacteria. Edman degradation confirmed that rCMO purified from the E. coli system and nCMO shared the same N-terminal sequence, whereas no sequence information could be obtained for rCMO expressed in yeast. Therefore, the yeast-expressed enzyme possesses an additional posttranslational modification(s), possibly acylation, at the N-terminus. Expression in E. coli is the preferred system for future site-directed mutagenesis studies and crystallization efforts.
Q-Index Code C1
Q-Index Status Provisional Code
Institutional Status Non-UQ

Document type: Journal Article
Sub-type: Article (original research)
Collection: School of Biomedical Sciences Publications
 
Versions
Version Filter Type
Citation counts: TR Web of Science Citation Count  Cited 29 times in Thomson Reuters Web of Science Article | Citations
Scopus Citation Count Cited 30 times in Scopus Article | Citations
Google Scholar Search Google Scholar
Created: Fri, 21 Oct 2011, 10:46:01 EST by Matthew Cheesman on behalf of School of Biomedical Sciences