Protein expression and purification, molecular interaction, and X-ray crystallographic analysis of baculovirus protein PK2.

Phosphorylation of the α subunit of eukaryotic translation initiation factor 2 (eIF2α) by eIF2α kinases is a common mechanism to regulate the initiation of translation under stress conditions. The PK2 protein from baculovirus Autographica californica multiple nucleopolyhedrovirus (AcMNPV) binds and inhibits eIF2α kinases to ensure efficient virus propagation. The C-terminal region of PK2 shares a homology with the C-lobe of eIF2α kinases, but the N-terminal region of PK2 is unique to the orthologous proteins. In order to understand the detailed structure and function of PK2, both the full-length PK2 and its N-terminal truncated protein (PK2Δ22) were expressed as a His-tag fusion protein in Escherichia coli and purified by three steps of chromatography. Notably, the cysteine mutant, PK2 C181S/C211S, promotes the solubility and stability of the PK2 protein. The results of the size-exclusion chromatography showed that the full-length PK2 exists in both multimeric and monomeric forms, and the molecular interaction of PK2 and the eIF2α kinase domain. The purified proteins were used further to screen various conditions to obtain these crystals. Crystals of the full-length PK2 and PK2Δ22 were obtained by a sitting-drop vapour-diffusion method using lithium sulfate and PEG3350 as the precipitant, respectively. The crystal of PK2 belonged to space group P41212, and diffracted X-rays to 2.7 Å resolution. The asymmetric unit contained four molecules of the protein, and the solvent content was 67.4%. Whereas, the crystal of the PK2Δ22 belonged to space group P212121, diffracted X-rays to 2.8 Å resolution. The asymmetric unit contained three molecules of the protein, and the solvent content was 48.1%. The crystallographic study of the PK2 protein will provide mechanistic insights into the inhibition of eIF2α kinase by the PK2 protein, and also pave the way for the improvement of the baculovirus-based protein expression system.