Endocrine Abstracts

11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1) has been implicated in the pathogenesis of human obesity and insulin resistance through 11-oxoreductase activation of cortisone (E) to cortisol (F) within the endoplasmic reticulum (ER) of adipocytes and hepatocytes. In its purified state 11beta-HSD1 is principally a dehydrogenase, converting F to E. Oxo-reductase activity can be regained by the addition of an NADPH regeneration system. In vivo hexose-6-phosphate dehydrogenase (H6PDH) directly determines the reaction direction of 11beta-HSD1 in intact cells by regenerating NADPH in the ER lumen (1,2).

The aim of our study is to optimise the production of recombinant homologous H6PDH protein, using bacterial expression systems, for future structure/function analyses. Recombinant H6PDH is largely insoluble, forming inclusion bodies, when expressed in E.coli.

We present an optimised protocol for the refolding and purification of human H6PDH from inclusion bodies. For bacterial expression a truncated H6PDH construct in pET28b(+) was transformed into E.coli strain BL21(DE3). Refolding of recombinant H6PDH protein from inclusion bodies was by rapid dilution. We examined the impact on refolding efficiency of the following variables; pH, temperature, redox conditions, and additives including aggregation suppressors, substrates and oxidising agents. Optimal refolding conditions were determined using sparse matrix and central composite experimental designs. The highest levels of soluble, active H6PDH protein were achieved by rapid dilution in 1M arginine, 0.1M Tris, 0.25M NaCl, 0.1 per cent Tween 60, pH8.0, with incubation overnight at 15degC. The refolded enzyme could then be purified by Ni-NTA metal affinity chromatography and gel filtration. Using these purification techniques we achieved a 48 fold purification of the enzyme, leading to specific activity values of 10.6 nmol/min/mg. The availability of purified recombinant H6PDH will now facilitate detailed kinetic and structural characterisation of the wild-type enzyme, and known mutant forms. (1) Nat Genet 2003, 34:434 (2) FEBS Letters 2004, 571:129.