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Optimization of expression and purification of acetoacetyl CoA thiolase from sunflower cotyledon
Author(s) -
Gomez Iris,
Schiedel Anke,
Oeljeklaus Silke,
Dyer James
Publication year - 2009
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.23.1_supplement.536.3
Subject(s) - thiolase , sunflower , affinity chromatography , chemistry , biochemistry , biology , chromatography , peroxisome , gene , enzyme , agronomy
Sunflower (Helianthus annus L) cotyledons have two thiolase activities which have been identified previously. Thiolase I (acetoacetyl CoA thiolase) has specificity for the four‐carbon acetoacetyl CoA, resulting in the production of two Acetyl CoA molecules. Thiolase II (oxoacyl CoA thiolase) is active towards short, medium and long chain acyl CoAs. The purpose of this research was to optimize the expression and purification of a Thiolase I from an E. coli expression system. The sunflower thiolase was cloned into the pBAD‐HisB expression vector, and this construct was transformed into BL‐21 E. coli which were induced for production of the Thiolase I with 0.2% arabinose. The soluble Thiolase 1 was purified using Ni‐NTA agarose affinity chromatography with a single band appearing on a Coomassie Blue stained SDS‐PAGE in the fraction eluted from the column with 100 mM imidazole.