Heterologous expression of human transketolase

Schenk, G, Duggleby, RG and Nixon, PF (1998) Heterologous expression of human transketolase. International Journal of Biochemistry and Cell Biology, 30 3: 369-378. doi:10.1016/S1357-2725(97)00154-4

Author Schenk, G
Duggleby, RG
Nixon, PF
Title Heterologous expression of human transketolase
Journal name International Journal of Biochemistry and Cell Biology   Check publisher's open access policy
ISSN 1357-2725
Publication date 1998-03-01
Year available 1998
Sub-type Article (original research)
DOI 10.1016/S1357-2725(97)00154-4
Open Access Status Not yet assessed
Volume 30
Issue 3
Start page 369
End page 378
Total pages 10
Place of publication OXFORD
Publisher Elsevier BV
Language eng
Subject 1303 Specialist Studies in Education
1307 Cell Biology
Abstract Transketolase belongs to the family of thiamin diphosphate dependent enzymes. 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-phosphate 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 these values were 85 +/- 16 nM and 0.74 +/- 0.23 mu M. The kinetic constants are similar to those of the native enzyme purified from human erythrocytes. Despite the modest expression level the reported system is well suited to a variety of functional studies. (C) 1998 Elsevier Science Ltd. All rights reserved.
Keyword Heterologous expression
Kinetic parameters
Protein purification
Recombinant human transketolase
Thiamin diphosphate
Q-Index Code C1
Q-Index Status Provisional Code
Institutional Status Unknown

Document type: Journal Article
Sub-type: Article (original research)
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