Cloning, expression, and purification of glyoxysomal 3-oxoacyl-CoA thiolase from sunflower cotyledons

Anke C. Schiedel, Silke Oeljeklaus, Patricia Minihan, Jim Dyer

Research output: Contribution to journalArticleResearchpeer-review

6 Citations (Scopus)

Abstract

The glyoxysomal β-oxidation system in sunflower (Helianthus annuus L.) cotyledons is distinguished by the coexistence of two different thiolase isoforms, thiolase I and II. So far, this phenomenon has only been described for glyoxysomes from sunflower cotyledons. Thiolase I (acetoacetyl-CoA thiolase, EC 2.3.1.9) recognizes acetoacetyl-CoA only, while thiolase II (3-oxoacyl-CoA thiolase, EC 2.3.1.16) exhibits a more broad substrate specificity towards 3-oxoacyl-CoA esters of different chain length. Here, we report on the cloning of thiolase II from sunflower cotyledons. The known DNA sequence of Cucumis sativus 3-oxoacyl-CoA thiolase was used to generate primers for cloning the corresponding thiolase from sunflower cotyledons. RT-PCR was then used to generate an internal fragment of the sunflower thiolase gene and the termini were isolated using 5′- and 3′-RACE. Full-length cDNA was generated using RT-PCR with sunflower thiolase-specific primers flanking the coding region. The resultant gene encodes a thiolase sharing at least 80% identity with other plant thiolases at the amino acid level. The recombinant sunflower thiolase II was expressed in a bacterial system in an active form and purified to apparent homogeneity in a single step using Ni-NTA agarose chromatography. The enzyme was purified 53.4-fold and had a specific activity of 235 nkat/mg protein. Pooled fractions from the Ni-NTA column resulted in an 83% yield of active enzyme to be used for further characterization.

Original languageEnglish
Pages (from-to)25-33
Number of pages9
JournalProtein Expression and Purification
Volume33
Issue number1
DOIs
StatePublished - 1 Jan 2004

Fingerprint

Acetyl-CoA C-Acyltransferase
Cotyledon
Helianthus
Organism Cloning
Glyoxysomes
Acetyl-CoA C-Acetyltransferase
Cucumis sativus
Polymerase Chain Reaction
Agarose Chromatography
Enzymes
Coenzyme A
Substrate Specificity
Genes
Protein Isoforms
Esters
Complementary DNA
Amino Acids

Keywords

  • Expression
  • Glyoxysomal thiolase
  • Purification
  • Sunflower

Cite this

Schiedel, Anke C. ; Oeljeklaus, Silke ; Minihan, Patricia ; Dyer, Jim. / Cloning, expression, and purification of glyoxysomal 3-oxoacyl-CoA thiolase from sunflower cotyledons. In: Protein Expression and Purification. 2004 ; Vol. 33, No. 1. pp. 25-33.
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abstract = "The glyoxysomal β-oxidation system in sunflower (Helianthus annuus L.) cotyledons is distinguished by the coexistence of two different thiolase isoforms, thiolase I and II. So far, this phenomenon has only been described for glyoxysomes from sunflower cotyledons. Thiolase I (acetoacetyl-CoA thiolase, EC 2.3.1.9) recognizes acetoacetyl-CoA only, while thiolase II (3-oxoacyl-CoA thiolase, EC 2.3.1.16) exhibits a more broad substrate specificity towards 3-oxoacyl-CoA esters of different chain length. Here, we report on the cloning of thiolase II from sunflower cotyledons. The known DNA sequence of Cucumis sativus 3-oxoacyl-CoA thiolase was used to generate primers for cloning the corresponding thiolase from sunflower cotyledons. RT-PCR was then used to generate an internal fragment of the sunflower thiolase gene and the termini were isolated using 5′- and 3′-RACE. Full-length cDNA was generated using RT-PCR with sunflower thiolase-specific primers flanking the coding region. The resultant gene encodes a thiolase sharing at least 80{\%} identity with other plant thiolases at the amino acid level. The recombinant sunflower thiolase II was expressed in a bacterial system in an active form and purified to apparent homogeneity in a single step using Ni-NTA agarose chromatography. The enzyme was purified 53.4-fold and had a specific activity of 235 nkat/mg protein. Pooled fractions from the Ni-NTA column resulted in an 83{\%} yield of active enzyme to be used for further characterization.",
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Cloning, expression, and purification of glyoxysomal 3-oxoacyl-CoA thiolase from sunflower cotyledons. / Schiedel, Anke C.; Oeljeklaus, Silke; Minihan, Patricia; Dyer, Jim.

In: Protein Expression and Purification, Vol. 33, No. 1, 01.01.2004, p. 25-33.

Research output: Contribution to journalArticleResearchpeer-review

TY - JOUR

T1 - Cloning, expression, and purification of glyoxysomal 3-oxoacyl-CoA thiolase from sunflower cotyledons

AU - Schiedel, Anke C.

AU - Oeljeklaus, Silke

AU - Minihan, Patricia

AU - Dyer, Jim

PY - 2004/1/1

Y1 - 2004/1/1

N2 - The glyoxysomal β-oxidation system in sunflower (Helianthus annuus L.) cotyledons is distinguished by the coexistence of two different thiolase isoforms, thiolase I and II. So far, this phenomenon has only been described for glyoxysomes from sunflower cotyledons. Thiolase I (acetoacetyl-CoA thiolase, EC 2.3.1.9) recognizes acetoacetyl-CoA only, while thiolase II (3-oxoacyl-CoA thiolase, EC 2.3.1.16) exhibits a more broad substrate specificity towards 3-oxoacyl-CoA esters of different chain length. Here, we report on the cloning of thiolase II from sunflower cotyledons. The known DNA sequence of Cucumis sativus 3-oxoacyl-CoA thiolase was used to generate primers for cloning the corresponding thiolase from sunflower cotyledons. RT-PCR was then used to generate an internal fragment of the sunflower thiolase gene and the termini were isolated using 5′- and 3′-RACE. Full-length cDNA was generated using RT-PCR with sunflower thiolase-specific primers flanking the coding region. The resultant gene encodes a thiolase sharing at least 80% identity with other plant thiolases at the amino acid level. The recombinant sunflower thiolase II was expressed in a bacterial system in an active form and purified to apparent homogeneity in a single step using Ni-NTA agarose chromatography. The enzyme was purified 53.4-fold and had a specific activity of 235 nkat/mg protein. Pooled fractions from the Ni-NTA column resulted in an 83% yield of active enzyme to be used for further characterization.

AB - The glyoxysomal β-oxidation system in sunflower (Helianthus annuus L.) cotyledons is distinguished by the coexistence of two different thiolase isoforms, thiolase I and II. So far, this phenomenon has only been described for glyoxysomes from sunflower cotyledons. Thiolase I (acetoacetyl-CoA thiolase, EC 2.3.1.9) recognizes acetoacetyl-CoA only, while thiolase II (3-oxoacyl-CoA thiolase, EC 2.3.1.16) exhibits a more broad substrate specificity towards 3-oxoacyl-CoA esters of different chain length. Here, we report on the cloning of thiolase II from sunflower cotyledons. The known DNA sequence of Cucumis sativus 3-oxoacyl-CoA thiolase was used to generate primers for cloning the corresponding thiolase from sunflower cotyledons. RT-PCR was then used to generate an internal fragment of the sunflower thiolase gene and the termini were isolated using 5′- and 3′-RACE. Full-length cDNA was generated using RT-PCR with sunflower thiolase-specific primers flanking the coding region. The resultant gene encodes a thiolase sharing at least 80% identity with other plant thiolases at the amino acid level. The recombinant sunflower thiolase II was expressed in a bacterial system in an active form and purified to apparent homogeneity in a single step using Ni-NTA agarose chromatography. The enzyme was purified 53.4-fold and had a specific activity of 235 nkat/mg protein. Pooled fractions from the Ni-NTA column resulted in an 83% yield of active enzyme to be used for further characterization.

KW - Expression

KW - Glyoxysomal thiolase

KW - Purification

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U2 - 10.1016/j.pep.2003.08.022

DO - 10.1016/j.pep.2003.08.022

M3 - Article

VL - 33

SP - 25

EP - 33

JO - Protein Expression and Purification

JF - Protein Expression and Purification

SN - 1046-5928

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