Expression and Automated Purification of Acetoacetyl CoA Thiolase from Sunflower Cotyledon

Jim Dyer, James Becker, Victor Geraldo, Mario Giron

Research output: Contribution to journalArticle

Abstract

In the sunflower (Helianthus annuus L.) cotyledons, two distinguishable thiolase activities have been identified: acetoacetyl CoA thiolase (AACT), specifically active during oxidation of short chain acetoacetyl CoA, and 3-oxoacyl CoA thiolase (OACT), active towards short, medium, and long-chain acyl CoAs. The purpose of this research was to optimize the purification of the AACT expressed in bacteria. Escherischia coli (E. coli) with the full-length sunflower AACT cDNA cloned into the expression vector pBAD-His B was induced for production of the AACT using 0.2% arabinose. Optimizing the conditions for protein purification is often an extremely empirical process requiring several iterations of a basic protocol each with specific changes. This iterative process was simplified using the programmable ã„kta chromatography system from Amersham Pharmacia (now GE Healthcare). Programming this system for automatic sample injection and variation of elution conditions allows for optimization of the protein purification protocol to be achieved overnight as opposed to taking a day for each iteration done manually. Once optimized, soluble AACT was purified from E. coli bacteria in one step using a His HiTrap HP column containing Ni-Sepharose resin in conjunction with the ã„kta chromatography system. AACT activity was detected in the induced bacteria, whereas no activity was observed with the uninduced control.

Original languageEnglish
Pages (from-to)85-87
Number of pages3
JournalJournal of Laboratory Automation
Volume11
Issue number2
DOIs
StatePublished - 1 Jan 2006

Fingerprint

Acetyl-CoA C-Acetyltransferase
Cotyledon
Helianthus
Purification
Bacteria
Chromatography
Proteins
Network protocols
Computer systems programming
Resins
Acetyl-CoA C-Acyltransferase
Oxidation
Arabinose
Sepharose
Complementary DNA
Delivery of Health Care
Injections
Research

Keywords

  • automation
  • glyoxysomal
  • protein purification
  • sunflower
  • thiolase

Cite this

Dyer, Jim ; Becker, James ; Geraldo, Victor ; Giron, Mario. / Expression and Automated Purification of Acetoacetyl CoA Thiolase from Sunflower Cotyledon. In: Journal of Laboratory Automation. 2006 ; Vol. 11, No. 2. pp. 85-87.
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Expression and Automated Purification of Acetoacetyl CoA Thiolase from Sunflower Cotyledon. / Dyer, Jim; Becker, James; Geraldo, Victor; Giron, Mario.

In: Journal of Laboratory Automation, Vol. 11, No. 2, 01.01.2006, p. 85-87.

Research output: Contribution to journalArticle

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