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Title: Purification, crystallization and preliminary X-ray diffraction analysis of the kinase domain of human tousled-like kinase 2

The C-terminal kinase domain of TLK2 (a human tousled-like kinase) has been cloned and overexpressed in Escherichia coli followed by purification to homogeneity. Crystallization experiments in the presence of ATP-γ-S yielded crystals suitable for X-ray diffraction analysis belonging to two different space groups: tetragonal I4{sub 1}22 and cubic P2{sub 1}3. Tousled-like kinases (TLKs) are an evolutionarily conserved family of serine/threonine protein kinases involved in chromatin dynamics, including DNA replication and repair, transcription and chromosome segregation. The two members of the family reported in humans, namely TLK1 and TLK2, localize to the cell nucleus and are capable of forming homo- or hetero-oligomers by themselves. To characterize the role of TLK2, its C-terminal kinase domain was cloned and overexpressed in Escherichia coli followed by purification to homogeneity. Crystallization experiments in the presence of ATP-γ-S yielded crystals suitable for X-ray diffraction analysis belonging to two different space groups: tetragonal I4{sub 1}22 and cubic P2{sub 1}3. The latter produced the best diffracting crystal (3.4 Å resolution using synchrotron radiation), with unit-cell parameters a = b = c = 126.05 Å, α = β = γ = 90°. The asymmetric unit contained one protein molecule, with a Matthews coefficient of 4.59 Å{sup 3} Da{sup −1}more » and a solvent content of 73.23%.« less
Authors:
;  [1] ;  [1] ;  [2] ;  [1]
  1. Spanish National Cancer Research Centre (CNIO), Melchor Fernández Almagro 3, 28029 Madrid (Spain)
  2. (Denmark)
Publication Date:
OSTI Identifier:
22375681
Resource Type:
Journal Article
Resource Relation:
Journal Name: Acta crystallographica. Section F, Structural biology communications; Journal Volume: 70; Journal Issue: Pt 3; Other Information: PMCID: PMC3944701; PMID: 24598926; PUBLISHER-ID: rl5059; OAI: oai:pubmedcentral.nih.gov:3944701; Copyright (c) Garrote et al. 2014; This is an open-access article distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original authors and source are cited.; Country of input: International Atomic Energy Agency (IAEA)
Country of Publication:
United States
Language:
English
Subject:
75 CONDENSED MATTER PHYSICS, SUPERCONDUCTIVITY AND SUPERFLUIDITY; CRYSTALLIZATION; CRYSTALS; ESCHERICHIA COLI; MOLECULES; RESOLUTION; SEGREGATION; SOLVENTS; SPACE GROUPS; SYNCHROTRON RADIATION; X-RAY DIFFRACTION