Cloning, expression and purification of an acetoacetyl CoA thiolase from sunflower cotyledon

James H. Dyer, Anthony Maina, Iris D. Gomez, Melissa Cadet, Silke Oeljeklaus, Anke C. Schiedel

Research output: Contribution to journalArticle

14 Citations (Scopus)

Abstract

Thiolase I and II coexist as part of the glyoxysomal β-oxidation system in sunflower (Helianthus annuus L.) cotyledons, the only system shown to have both forms. The importance of thiolases can be underscored not only by their ubiquity, but also by their involvement in a wide variety of processes in plants, animals and bacteria. Here we describe the cloning, expression and purification of acetoacetyl CoA thiolase (AACT) in enzymatically active form. Use of the extensive amount of sequence information from the databases facilitated the efficient generation of the gene-specific primers used in the RACE protocols. The recombinant AACT (1233 bp) shares 75% similarity with other plant AACTs. Comparison of specific activity of this recombinant AACT to a previously reported enzyme purified from primary sunflower cotyledon tissue was very similar (263 nkat/mg protein vs 220 nkat/mg protein, respectively). Combining the most pure fractions from the affinity column, the enzyme was purified 88-fold with a 55% yield of the enzymatically active, 47 kDa AACT.

Original languageEnglish
Pages (from-to)736-744
Number of pages9
JournalInternational Journal of Biological Sciences
Volume5
Issue number7
DOIs
StatePublished - 1 Jan 2009

Fingerprint

acetyl-CoA acetyltransferase
Acetyl-CoA C-Acetyltransferase
Cotyledon
Helianthus
Helianthus annuus
purification
cotyledons
Organism Cloning
molecular cloning
enzyme
protein
fold
oxidation
bacterium
gene
animal
Enzymes
enzymes
Proteins
proteins

Keywords

  • Acetoacetyl CoA
  • Cloning
  • Expression
  • Sunflower
  • Thiolase

Cite this

Dyer, James H. ; Maina, Anthony ; Gomez, Iris D. ; Cadet, Melissa ; Oeljeklaus, Silke ; Schiedel, Anke C. / Cloning, expression and purification of an acetoacetyl CoA thiolase from sunflower cotyledon. In: International Journal of Biological Sciences. 2009 ; Vol. 5, No. 7. pp. 736-744.
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Cloning, expression and purification of an acetoacetyl CoA thiolase from sunflower cotyledon. / Dyer, James H.; Maina, Anthony; Gomez, Iris D.; Cadet, Melissa; Oeljeklaus, Silke; Schiedel, Anke C.

In: International Journal of Biological Sciences, Vol. 5, No. 7, 01.01.2009, p. 736-744.

Research output: Contribution to journalArticle

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