title: Cloning and characterization of GST fusion tag stabilized large subunit of Escherichia coli acetohydroxyacid synthase I.
abstract: There are three acetohydroxyacid synthase (AHAS, EC 4.1.3.18) isozymes (I, II, and III) in the enterobacteria Escherichia coli among which AHAS I is the most active. Its large subunit (LSU) possesses full catalytic machinery, but is unstable in the absence of the small subunit (SSU). To get applicable LSU of AHAS I, we prepared and characterized in this study the polypeptide as a His-tagged (His-LSU) and a glutathione S-transferase (GST)-tagged (GST-LSU) fusion protein, respectively. The results showed that the His-LSU is unstable, whereas the GST-LSU displays excellent stability. This phenomenon suggests that the GST polypeptide fusion tag could stabilize the target protein when compared with histidine tag. It is the first time that the stabilizing effect of the GST tag was observed. Further characterization of the GST-LSU protein indicated that it possesses the basic functions of AHAS I with a specific activity of 20.8 μmol min(-1) mg(-1) and a Km value for pyruvate of 0.95 mM. These observations imply that introduction of the GST fusion tag to LSU of AHAS I does not affect the function of the protein. The possible reasons that the GST fusion tag could make the LSU stable are initially discussed.
authors.affiliation: College of Life Sciences, Northwest University, 229 North Taibai Road, Xi'an, Shaanxi 710069, PR China.
authors.forename: Heng
authors.initials: H
authors.lastname: Li
authors.affiliation: College of Life Sciences, Northwest University, 229 North Taibai Road, Xi'an, Shaanxi 710069, PR China.
authors.forename: Nan
authors.initials: N
authors.lastname: Liu
authors.affiliation: College of Life Sciences, Northwest University, 229 North Taibai Road, Xi'an, Shaanxi 710069, PR China.
authors.forename: Wen-Ting
authors.initials: WT
authors.lastname: Wang
authors.affiliation: College of Life Sciences, Northwest University, 229 North Taibai Road, Xi'an, Shaanxi 710069, PR China.
authors.forename: Ji-Yu
authors.initials: JY
authors.lastname: Wang
authors.affiliation: College of Life Sciences, Northwest University, 229 North Taibai Road, Xi'an, Shaanxi 710069, PR China. Electronic address: gaowenyun@nwu.edu.cn.
authors.forename: Wen-Yun
authors.initials: WY
authors.lastname: Gao
mesh_terms: D000095:Acetolactate Synthase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D000595:Amino Acid Sequence; D001547:Benzaldehydes / Q000378:metabolism; D003001:Cloning, Molecular; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D005982:Glutathione Transferase / Q000235:genetics / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D019289:Pyruvic Acid / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013696:Temperature
chemical_list: D001547:Benzaldehydes; D029968:Escherichia coli Proteins; D021122:Protein Subunits; D011993:Recombinant Fusion Proteins; D019289:Pyruvic Acid; D000095:Acetolactate Synthase; D005982:Glutathione Transferase; C032175:benzaldehyde
keywords: Acetohydroxyacid synthase I; Characterization; Escherichia coli; Glutathione S-transferase fusion tag; Large subunit; Stability