Expression, purification from inclusion bodies, and crystal characterization of a transition state analog complex of arginine kinase: A model for studying phosphagen kinases

Abstract

Phosphagen kinases catalyze the reversible transfer of a phosphoryl group between guanidino phosphate compounds and ADP, thereby regenerating ATP during bursts of cellular activity. Large quantities of highly pure arginine kinase (EC 2.7.3.3), the phosphagen kinase present in arthropods, have been isolated from E. coli, into which the cDNA for the horseshoe crab enzyme had been cloned. Purification involves size exclusion and anion exchange chromatographies applied in the denatured and refolded states. The recombinant enzyme has been crystallized as a transition state analog complex. Near complete native diffraction data have been collected to 1.86 A resolution. Substitution of a recombinant source for a natural one, improvement in the purification, and data collection at cry0 temperatures have all yielded significant improvements in diffraction. This reaction and those of the rest of the guanidino (or phosphagen) kinase family, including creatine kinase (CK), enable ATP to be quickly regenerated from storage phosphagens during bursts of cellular activity. In addition to this "temporal" buffering, it has been proposed that these enzymes also function in "spatial" buff-

Transition state structure of arginine kinase: Implications for catalysis of bimolecular reactions

“…This finding was consistent with evidence for a conformational change that occurs when all substrates have been bound to either AK or CK (19,20). By contrast, the AK was expected to be in the ''closed'' reactive conformation because it was crystallized in the presence of the complete set of transition-state analog components (21).…”

“…AK from horseshoe crab (Limulus polyphemus) was cloned, expressed, complexed with analog components (ADP, nitrate, and arginine), as described (21,22). Diffraction data were collected with an R-Axis II image plate detector (Molecular Structure) and a rotating anode x-ray source for the previously reported crystals and a second crystal form crystallizing under identical conditions (Table 1).…”

Arginine kinase from Litopenaeus vannamei: Cloning, expression and catalytic properties

“…AK cDNA or protein has been obtained from a variety of crustaceans including the crab Carcinus maenas and Callinectes sapidus [3,4], shrimp Marsupenaeus japonicus [5], Penaeus aztecus [6], Penaeus monodon [7], Fenneropenaeus chinensis [8], Litopenaeus vannamei [9] etc. Some structural and catalytic characterizations of AK in crustaceans have also been reported [10][11][12][13][14].…”

A DNA vaccine targeting p42.3 induces protective antitumor immunity via eliciting cytotoxic CD8+T lymphocytes in a murine melanoma model

“…Subcloning of plasmid pET28a-p42.3 was performed in Dr. Lu's lab. Protein p42.3 was purified from pET28a-p42.3 transformed E. coli BL21 (DE3) according to a previous protocol 33 …”