High-level expression and purification of human thymidine kinase 1: quaternary structure, stability, and kinetics.
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 His(6)-tagged fusion proteins. Expression, isolation, and purification protocols have been established, leading to high yields of soluble and active wild type (approximately 35 mg) and truncated hTK1 (approximately 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-MgCl(2) 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 (T(m)) of 71.7 and 73.4 degrees C, respectively, when measured without adding substrates. The presence of substrates stabilized both hTK1W (DeltaT(m) ranging from 5.6 to 12.5 degrees C) and hTK1M (DeltaT(m) ranging from 0.8 to 5.3 degrees C). Both enzymes show high activity over a broad range of pH, temperature, and ionic strength. Kinetic studies determined a K(M) of 0.51 microM and a k(cat) of 0.28 s(-1) for wild-type hTK1. The truncated hTK1M has a K(M) of 0.87 microM and k(cat) of 1.65 s(-1), thus exhibiting increased catalytic efficiency. The availability of recombinant human TK1 will facilitate further biochemical and crystallographic studies.
Birringer
Markus S
MS
Institute of Pharmaceutical Sciences, Department of Chemistry and Applied Biosciences, Swiss Federal Institute of Technology (ETH), Zurich, Switzerland.
Perozzo
Remo
R
Kut
Elvan
E
Stillhart
Corinne
C
Surber
Wanda
W
Scapozza
Leonardo
L
Folkers
Gerd
G
D000595:Amino Acid Sequence; D018360:Crystallography, X-Ray; D004795:Enzyme Stability; D004926:Escherichia coli; D029968:Escherichia coli Proteins / Q000737:chemistry; D018840:HSP70 Heat-Shock Proteins / Q000737:chemistry; D006358:Hot Temperature; D006801:Humans; D007700:Kinetics; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification*; D017384:Sequence Deletion / Q000235:genetics; D013379:Substrate Specificity; D013936:Thymidine / Q000737:chemistry / Q000378:metabolism; D013937:Thymidine Kinase / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification*
D029968:Escherichia coli Proteins; D018840:HSP70 Heat-Shock Proteins; D011994:Recombinant Proteins; D013937:Thymidine Kinase; C092717:thymidine kinase 1; C063842:dnaK protein, E coli; D013936:Thymidine