Journal
PROTEIN EXPRESSION AND PURIFICATION
Volume 21, Issue 1, Pages 81-86Publisher
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1006/prep.2000.1340
Keywords
-
Categories
Funding
- NIEHS NIH HHS [ES05780] Funding Source: Medline
- NIGMS NIH HHS [GM07750, GM43511] Funding Source: Medline
Ask authors/readers for more resources
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 His(6)-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 His(6)-tagged rCMO and nCMO toward their index substrate, cyclohexanone, were similar and ranged from 14 to 20 mu mol/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 His(6)-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. (C) 2001 Academic Press.
Authors
I am an author on this paper
Click your name to claim this paper and add it to your profile.
Reviews
Recommended
No Data Available