Optimization of expression and properties of the recombinant acetohydroxyacid synthase of Thermotoga maritima

Abstract

The data provide additional support of the characterization of the biophysical and biochemical properties of the enzyme acetohydroxyacid synthase from the hyperthermophilic bacterium Thermotoga maritima (Eram et al., 2015) [1]. The genes encoding the enzyme subunits have been cloned and expressed in the mesophilic host Escherichia coli. Detailed data include information about the optimization of the expression conditions, biophysical properties of the enzyme and reconstitution of the holoenzyme from individually expressed and purified subunits.

Characterization of acetohydroxyacid synthase from the hyperthermophilic bacterium Thermotoga maritima

“…strain RQ2 (GenBank accession number NC_010483) (see Ref. [46, Table 1] ). Only the genome of Thermotoga lettingae (GenBank accession number NC_009828) had no recognizable ilv operon, which is in accordance with organism’s auxotrophy for leucine, valine, and isoleucine [47] .…”

“…In the case of the construct pETTm0548/9, only the catalytic subunit was expressed as a soluble protein, though the yield of the regulatory subunit was very low, suggesting the possible proteolysis or incomplete translation of the subunit (Ref. [46, Fig. 1] ).…”

“…During heat-treatment, majority of mesophilic host proteins are denatured and aggregated, rendering the recombinant thermostable protein in soluble fraction [48] (see Ref. [46, Table 2] ).…”

“…The effects of growth temperature (Ref. [46, Figs. 2–4] ) and heat-treatment temperatures on yield of the soluble protein and enzyme activity were also optimized (Ref.…”

Pyruvate decarboxylase activity of the acetohydroxyacid synthase of Thermotoga maritima

“…The recombinant plasmids for the expression of genes encoding the catalytic and regulatory subunits were constructed and propagated in Escherichia coli strain DH5α. The recombinant proteins overexpressed under the control of T7 polymerase of the plasmid pET30a (+) (Novagen, WI, USA) in E. coli BL21 (DE3) Rosetta 2 [F − ompT hsdS B (rB − mB − ) gal dcm pRARE27 ( CamR )] (Novagen, WI, USA) as described elsewhere [10] , [11] . The recombinant catalytic subunit was purified by heat-induced precipitation of the host proteins (1 h at 80 °C) followed by DEAE and HAP column chromatography as described previously [10] , [11] .…”