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

Genfa Zhou, Gopalakrishnan Parthasarathy, Thayumanasamy Somasundaram, Andrea Ables, Lance Roy, Scott J. Strong, W. Ross Ellington, Michael S. Chapman

Research output: Contribution to journalArticle

20 Scopus citations

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 onion 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.

Original languageEnglish (US)
Pages (from-to)444-449
Number of pages6
JournalProtein Science
Volume6
Issue number2
DOIs
StatePublished - Feb 1997

Keywords

  • arginine kinase
  • crystals
  • expression
  • guanidino kinase
  • high resolution
  • phosphagen kinase
  • transition state complex

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology

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