Journal of Microbiology and Biotechnology

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
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Purification and Characterization of the Functional Catalytic Domain of PKR-Like Endoplasmic Reticulum Kinase Expressed in Escherichia coli

  • Yun Jin-A (Department of Biotechnology, Yonsei University) ;
  • Chung Ho-Young (Department of Biotechnology, Yonsei University) ;
  • Kim Seong-Jun (Department of Biotechnology, Yonsei University) ;
  • Cho Hyun-Soo (Department of Biology, Yonsei University) ;
  • Oh Jong-Won (Department of Biotechnology, Yonsei University)
  • Published : 2006.09.01
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Abstract

PKR-like endoplasmic reticulum (ER) kinase (PERK) is a type I transmembrane ER-resident protein containing a cytoplasmic catalytic domain with a Ser/Thr kinase activity, which is most closely related to the eukaryotic translation initiation factor-$2{\alpha}$ ($eIF2{\alpha}$) kinase PKR involved in the antiviral defense pathway by interferon. We cloned and expressed the PERK C-terminal kinase domain (cPERK) in Escherichia coli. Like PERK activation in cells under ER stress, wild-type cPERK underwent autophosphorylation when overexpressed in E. coli, whereas the cPERK(K621M) with a methionine substitution for the lysine at amino acid 621 lost the autophosphorylation activity. The activated form cPERK which was purified to near homogeneity, formed an oligomer and was able to trans-phosphorylate specifically its cellular substrate $eIF2{\alpha}$. Two-dimensional phosphoamino acids analysis revealed that phosphorylation of cPERK occurs at the Ser and Thr residues. The functionally active recombinant cPERK, and its inactive mutant should be useful for the analysis of biochemical functions of PERK and for the determination of their three-dimensional structures.

Keywords

References

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