Expression, mutagenesis and characterization of E. coli acetohydroxyacid synthase II

Hill, Craig (1998). Expression, mutagenesis and characterization of E. coli acetohydroxyacid synthase II PhD Thesis, School of Molecular and Microbial Sciences, University of Queensland.

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Author Hill, Craig
Thesis Title Expression, mutagenesis and characterization of E. coli acetohydroxyacid synthase II
School, Centre or Institute School of Molecular and Microbial Sciences
Institution University of Queensland
Publication date 1998-01-01
Thesis type PhD Thesis
Supervisor R. G. Duggleby
Total pages 244
Language eng
Subjects 0605 Microbiology
Formatted abstract
Several classes of herbicide act by inhibiting the enzyme acetohydroxyacid synthase. This report presents the results of a study on acetohydroxyacid synthase isozyme II from Escherichia coli and the interaction of the wild-type and mutant forms of the enzyme with a number of herbicides. 

Acetohydroxyacid synthase has a low abundance in native sources and is often poorly expressed in recombinant systems. In order to produce sufficient enzyme for characterization, the genes encoding the two subunits of acetohydroxyacid synthase II were subcloned into a series of expression vectors. Expression of the enzyme in its native form from plasmids with different features such as low and high copy number and lac, tac and T7 promoters all resulted in relatively low yields of the enzyme. The highest level of expression achieved for the native form of the enzyme corresponds to 2.4% of the protein in the soluble cell extract where a specific activity of 0.58 U/mg was observed. Expression of acetohydroxyacid synthase II as a GST-fusion protein resulted in a large increase in the abundance of the enzyme. However the enzyme was produced in an insoluble form. Lowering the induction temperature did not result in expression of soluble protein. Attempts to solubilize the enzyme using detergents were also unsuccessful. Replacing the GST-fusion peptide with the smaller one encoded by the pET30(a)+ vector also conferred a high level of expression on acetohydroxyacid synthase II. The solubility of the enzyme expressed from this construct was temperature sensitive; induction at 30°C gave soluble enzyme whereas at 37°C the protein fractionated with the insoluble material. The abundance of the fusion enzyme expressed from the pET30(a)+ vector is the highest reported for any acetohydroxyacid synthase and constitutes approximately 30% of the protein in the soluble cell extract. A specific activity of 14 U/mg was observed in this fraction. 

The N-terminal fusion of proteins expressed from the pET30(a)+ vector contain an oligohistidine domain that allows a single step purification by immobilized metal affinity chromatography. Employing this method of purification gave a pure preparation of fusion enzyme with a specific activity of 53 U/mg that is the highest reported for an acetohydroxyacid synthase from any source. Surprisingly the native form of acetohydroxyacid synthase II also absorbs to the metal affinity column. This step followed by blue dye chromatography gave an apparently pure native enzyme with a specific activity of 24 U/mg. ...............................
Keyword Escherichia coli
Additional Notes Variant title: Expression, mutagenesis and charterization of E. coli acetohydroxyacid synthase II

Document type: Thesis
Collection: UQ Theses (RHD) - UQ staff and students only
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