Cloning and functional analysis of PKZ (PKR-like) from grass carp (Ctenopharyngodon idellus)

    loading  Checking for direct PDF access through Ovid

Abstract

The new teleost fish PKZ (PKR-like) full-length cDNA (GU299765) had been cloned and identified from grass carp (Ctenopharyngodon idellus). The cDNA of grass carp PKZ (CiPKZ) has 2185 bp in length with a largest open reading frame (ORF) encoding 513aa. CiPKZ possesses a conserved C-terminal catalytic domain of eIF2α kinase family. Within its N-terminal there are two binding domain (Zα) named Zα1 (1–67aa) and Zα2 (81–152aa). BLAST homologous search reveals that CiPKZ has a high-level homology with other fish PKZs and PKRs. Like other fish PKZs and PKRs, CiPKZ is a ubiquitous tissue expression gene that had a very low level of constitutive expression but up-regulated in response to Poly I:C or hot stress (34 °C). For the purpose of searching for the potential function of CiPKZ, we obtained CiPKZ polypeptide via Escherichia coli Rosetta prokaryotic expression and purified with Ni-NTA His-Bind Resin affinity chromatography. CiPKZ polypeptide was used for the test of phosphorylating eIF2α in vitro. The results demonstrated that CiPKZ could be activated by Z-DNA but not by Poly I:C, and with subsequent could phosphorylate eIF2α. Meanwhile, four pcDNA3.1/PKZ recombinant plasmids, including pcDNA3.1/PKZ-wet, pcDNA3.1/PKZ-wet-K198R, pcDNA3.1/PKZ-wet-C, pcDNA3.1/PKZ-wet-C-K198R had been constructed, respectively. Mouse Myeloma cells (Sp2/0) and Human Umbilical Vein Endothelial Cells (HUVEC) were transiently cotransfected with pcDNA3.1/PKZ recombinant plasmid and PGL-3-promoter plasmid. The results revealed that CiPKZ could greatly decrease luciferase level in these cells. Zα and the K198 amino acid residue may play a key role in its function.

Related Topics

    loading  Loading Related Articles