Molecular cloning, expression and characterization of eukaryotic translation initiation factor 2α (eIF2α) kinase and eIF2α from Indian midge Chironomus ramosus

Background

Protein synthesis regulation via eukaryotic initiation factor 2α (eIF2α) phosphorylation is a conserved mechanism in eukaryotes that plays a vital role in cellular homeostasis and adaptation to stress conditions. Among eIF2α kinases, heme regulated inhibitor (HRI), protein kinase R (PKR), PKR like endoplasmic reticulum kinase (PERK), and general control non-derepressible 2 kinase (GCN2) mediate stress-induced translational control across various insect species. Despite extensive studies in insects such as Drosophila melanogaster and Bombyx mori, no attempts have been made to identify eIF2α kinases in aquatic insects, leaving a gap in understanding stress-regulated protein synthesis mechanisms in such organisms.

Objective

This study aimed to characterize a novel eIF2α kinase and its substrate, eIF2α from Chironomus ramosus, an Indian tropical midge known for its exceptional environmental stress tolerance.

Materials and methods

C. ramosus cultures were maintained under controlled laboratory conditions, and total RNA was extracted from the larvae for cDNA synthesis. Using degenerate primers, polymerase chain reaction (PCR) amplification was performed, followed by cloning into pCR®4-TOPO and subcloning into pET28a for expression studies in Escherichia coli (E. coli) BL21-Rosetta. Clones were validated via restriction digestion and DNA sequencing. Expression and purification of recombinant proteins were confirmed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis.

Results

PCR amplification yielded 2084 bp C. ramosus eIF2α kinase (CR-eIF2AK) and 1121 bp C. ramosus eIF2 α (CR-eIF2α) fragments, which have been successfully cloned and expressed. Western blot analysis confirmed the expression of ~79 kDa CR-eIF2AK and ~45 kDa CR-eIF2α proteins, establishing correct reading frame alignment. Phylogenetic analysis revealed its close homology with known eIF2α kinases, supporting its role in stress-induced translational control.

Conclusion

This is the first sequence-based characterization of eIF2α kinase and eIF2α from C. ramosus, contributing to novel insights into the molecular mechanisms regulating protein synthesis in aquatic insects.