HETEROLOGOUS EXPRESSION OF HUMAN TRANSKETOLASE

Transketolase belongs to the family of thiamin diphosphate dependent e nzymes. The aim of this study was to establish a bacterial expression system for human transketolase in order to investigate the functional characteristics of mammalian transketolases. The level of recombinant human enzyme expressed in Escherichia coli was modest. Purification of recombinant transketolase and separation from the E. coli enzyme has been greatly simplified by means of a non-cleavable hexa-histidine tag . The highest specific activity was 13.5 U/mg and the K-m values were 0.27 +/- 0.02 and 0.51 +/- 0.05 mM for the substrates D-xylulose 5-pho sphate and D-ribose 5-phosphate, respectively. Binding of cofactors to the apoenzyme showed the expected hysteresis. Without preincubation, the K-m values for thiamin diphosphate and for Mg2+ were, respectively , 4.1 +/- 0.8 and 2.5 +/- 0.4 mu M, but after 1 h of preincubation the se values were 85 +/- 16 nM and 0.74 +/- 0.23 mu M. The kinetic consta nts are similar to those of the native enzyme purified from human eryt hrocytes. Despite the modest expression level the reported system is w ell suited to a variety of functional studies. (C) 1998 Elsevier Scien ce Ltd. All rights reserved.