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Expression and Purification of Acetoacetyl CoA thiolase from Sunflower Cotyledon
Author(s) -
Giron Mario,
Maina Anthony,
Dyer James H.
Publication year - 2006
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.20.5.a948-d
Subject(s) - thiolase , sunflower , helianthus annuus , recombinant dna , affinity chromatography , biochemistry , enzyme , chemistry , biology , gene , peroxisome , agronomy
Thiolase activity isolated from sunflower (Helianthus annuus L.) cotyledons occurs as two forms. Thiolase I (AACT) shows activity only toward short chain acetoacetyl CoA, while thiolase II (OACT) exhibits activity toward both short and long‐chain acetoacetyl CoA and 3‐oxoacyl CoAs. We have previously cloned both AACT and OACT, and the full‐length thiolases were cloned into the bacterial expression vector pBAD‐HisB, expressed in E. coli and purified using Ni‐NTA affinity chromatography. The purpose of this research was to optimize conditions for expression and purification. Preliminary work was done to determine biochemical characteristics of the recombinant enzymes. Further work to determine additional biochemical characteristics of both AACT and OACT is underway, as well as production of sufficiently pure protein for crystallization.