Large-scale purification and characterization of recombinant Pseudomonas ceramidase: Regulation by calcium

Bill X. Wu, Christopher F. Snook, Motohiro Tani, Erika E. Büllesbach, Yusuf A. Hannun

Research output: Contribution to journalArticle

16 Citations (Scopus)

Abstract

Ceramidases (CDases) hydrolyze ceramide to sphingosine (SPH) and fatty acid. Pseudomonas CDase (pCDase) is a homolog of mammalian neutral ceramidases and may play roles in disease pathogenesis. In this study, pCDase was cloned and expressed in Escherichia coli (E. coli). The expressed recombinant pCDase was solubilized by optimizing several factors, including culture medium, the concentration of isopropyl-β-thiogalactopyranoside (IPTG), temperature, and time of induction, which were identified to be critical for the optimal production of recombinant pCDase. The recombinant pCDase was purified using nickel-nitrilotriacetic acid affinity, phenyl-Sepharose, and Q-Sepharose column chromatography, which gave an overall yield of 0.45 mg/l purified protein of starting culture. The activity of the recombinant pCDase followed classical Michaelis-Menten kinetics, with optimum activity in the neutral pH range. Both the hydrolytic and the reverse activities of CDase were stimulated by calcium with an affinity constant (Ka) of 1.5 μM. Kinetics studies showed that calcium caused a decrease of Km and an increase in V max of pCDase. Calcium and D-erythro-sphingosine caused significant changes in the near ultraviolet circular dichroism (CD) spectra and the changes were inhibited in the presence of EGTA. These results identify important interactions between calcium and pCDase, which may play an essential role in the interaction of pCDase and its substrate.

Original languageEnglish
Pages (from-to)600-608
Number of pages9
JournalJournal of Lipid Research
Volume48
Issue number3
DOIs
Publication statusPublished - Mar 1 2007
Externally publishedYes

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Ceramidases
Pseudomonas
Purification
Calcium
Neutral Ceramidase
Thiogalactosides
Column chromatography
Sphingosine
Kinetics
Ceramides
Egtazic Acid
Sepharose
Escherichia coli
Culture Media
Fatty Acids
Agarose Chromatography
Substrates
Circular Dichroism
Proteins

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Endocrinology
  • Cell Biology

Cite this

Large-scale purification and characterization of recombinant Pseudomonas ceramidase : Regulation by calcium. / Wu, Bill X.; Snook, Christopher F.; Tani, Motohiro; Büllesbach, Erika E.; Hannun, Yusuf A.

In: Journal of Lipid Research, Vol. 48, No. 3, 01.03.2007, p. 600-608.

Research output: Contribution to journalArticle

Wu, Bill X. ; Snook, Christopher F. ; Tani, Motohiro ; Büllesbach, Erika E. ; Hannun, Yusuf A. / Large-scale purification and characterization of recombinant Pseudomonas ceramidase : Regulation by calcium. In: Journal of Lipid Research. 2007 ; Vol. 48, No. 3. pp. 600-608.
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