en
Scientific article
English

High-level expression and purification of human thymidine kinase 1: Quaternary structure, stability, and kinetics

Published inProtein expression and purification, vol. 47, no. 2, p. 506-515
Publication date2006-06
Abstract

Human cytosolic thymidine kinase (hTK1) is the key enzyme of the pyrimidine salvage pathway and phosphorylates thymidine to thymidine monophosphate, a precursor building block of the DNA. Wild-type hTK1 (hTK1W) as well as a truncated form of the enzyme (hTK1M) carrying deletions at the N- and C-terminal regions were cloned as His6-tagged fusion proteins. Expression, isolation, and purification protocols have been established, leading to high yields of soluble and active wild type (∼35 mg) and truncated hTK1 (∼23 mg) per liter of culture. The protein was purified to near homogeneity. The chaperone DnaK was identified to be the major contaminant that could be removed by applying an additional ATP–MgCl2 incubation and washing step. hTK1W was a permanent tetramer in solution, whereas the truncated construct hTK1M appears to be a dimer in absence and presence of substrates. Both hTK1W and hTK1M exhibit pronounced thermal stability with transition temperatures (Tm) of 71.7 and 73.4 °C, respectively, when measured without adding substrates. The presence of substrates stabilized both hTK1W (ΔTm ranging from 5.6 to 12.5 °C) and hTK1M (ΔTm ranging from 0.8 to 5.3 °C). Both enzymes show high activity over a broad range of pH, temperature, and ionic strength. Kinetic studies determined a KM of 0.51 μM and a kcat of 0.28 s−1 for wild-type hTK1. The truncated hTK1M has a KM of 0.87 μM and kcat of 1.65 s−1, thus exhibiting increased catalytic efficiency. The availability of recombinant human TK1 will facilitate further biochemical and crystallographic studies.

eng
Keywords
  • Human thymidine kinase 1
  • Expression
  • Purification
  • Kinetics
  • Quaternary structure
  • CD spectroscopy
Citation (ISO format)
BIRRINGER, Markus S. et al. High-level expression and purification of human thymidine kinase 1: Quaternary structure, stability, and kinetics. In: Protein expression and purification, 2006, vol. 47, n° 2, p. 506–515. doi: 10.1016/j.pep.2006.01.001
Main files (1)
Article (Published version)
accessLevelRestricted
Identifiers
ISSN of the journal1046-5928
10views
0downloads

Technical informations

Creation01/02/2024 9:59:54 AM
First validation02/09/2024 11:33:24 AM
Update time02/09/2024 11:33:24 AM
Status update02/09/2024 11:33:24 AM
Last indexation02/09/2024 11:33:25 AM
All rights reserved by Archive ouverte UNIGE and the University of GenevaunigeBlack