Cloning, expression, characterization and phylogenetic analysis of arginine kinase from greasyback shrimp (Metapenaeus ensis)

摘要

Arginine kinase (AK) plays an important role in cellular energy metabolism in invertebrate. The encoding AK gene from Shrimp Metapenaeus ensis (M. ensis) was cloned in prokaryotic expression plasmid pET-28a, and it was then expressed in Escherichia coil in dissoluble form. The recombinant protein was purified by following three chromatography steps in turn: CM-Cellulose cation-exchange, Sephacryl S-100HR gel filtrate and DEAE-Sepharose anion-exchange. The purified AK’s apparent K_m was 2.33±0.1 and 1.59±0.2 mM for ATP and L-arginine, respectively, while its optimum pH and temperature was 8.5 and 30 °C in the process of forward reaction, respectively. Phylogenetic analysis of cDNA-derived amino acid sequences for the AKs indicated a close affinity of M. ensis and another shrimp (Litopenaeus vannamei).