Premium
Expression, purification from inclusion bodies, and crystal characterization of a transition state analog complex of arginine kinase: A model for studying phosphagen kinases
Author(s) -
Zhou Genfa,
Parthasarathy Gopalakrishnan,
Somasundaram Thayumanasamy,
Ables Andrea,
Roy Lance,
Chapman Michael S.,
Strong Scott J.,
Ellington W. Ross
Publication year - 1997
Publication title -
protein science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.353
H-Index - 175
eISSN - 1469-896X
pISSN - 0961-8368
DOI - 10.1002/pro.5560060222
Subject(s) - arginine kinase , kinase , biochemistry , transition state analog , recombinant dna , enzyme , biology , complementary dna , transferase , chemistry , arginine , amino acid , active site , gene
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 Å resolution. Substitution of a recombinant source for a natural one, improvement in the purification, and data collection at cryo temperatures have all yielded significant improvements in diffraction.