A single step purification for recombinant proteins. Characterization of a microtubule associated protein (MAP 2) fragment which associates with the type II cAMP-dependent protein kinase

FEBS Lett. 1992 May 18;302(3):274-8. doi: 10.1016/0014-5793(92)80458-s.

Abstract

A 167 base pair DNA cassette has been constructed to facilitate the detection and purification of recombinant proteins. This cassette, kfc, encodes three distinct peptide units: a phosphorylation site for the cAMP-dependent protein kinase (PKA), called kemptide, a factor Xa cleavage site, and a calmodulin-binding peptide. Expressed kfc fusion proteins can be purified from bacterial lysates in one step by affinity chromatography on calmodulin-agarose using EGTA as eluant. As a test of this system, we describe the expression, purification and characterization of the PKA binding domain of the microtubule associated protein (MAP 2).

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Base Sequence
  • Binding Sites
  • Calmodulin / metabolism
  • Calmodulin-Binding Proteins / genetics*
  • Cyclic AMP / pharmacology*
  • DNA / chemistry
  • Escherichia coli / genetics
  • Factor Xa / metabolism
  • Microtubule-Associated Proteins / genetics
  • Microtubule-Associated Proteins / isolation & purification*
  • Microtubule-Associated Proteins / metabolism
  • Molecular Sequence Data
  • Oligopeptides / metabolism
  • Peptide Fragments / chemistry
  • Peptide Fragments / genetics
  • Peptide Fragments / isolation & purification*
  • Phosphorylation
  • Protein Kinases / metabolism*
  • Protein-Serine-Threonine Kinases*
  • Recombinant Fusion Proteins / chemistry
  • Recombinant Fusion Proteins / genetics*
  • Recombinant Fusion Proteins / isolation & purification*
  • Transformation, Bacterial

Substances

  • Calmodulin
  • Calmodulin-Binding Proteins
  • Microtubule-Associated Proteins
  • Oligopeptides
  • Peptide Fragments
  • Recombinant Fusion Proteins
  • KFC protein, Gallus gallus
  • kemptide
  • DNA
  • Cyclic AMP
  • Protein Kinases
  • Protein-Serine-Threonine Kinases
  • Factor Xa

Associated data

  • GENBANK/S86390
  • GENBANK/S86392
  • GENBANK/S90294
  • GENBANK/S90296
  • GENBANK/S90298
  • GENBANK/X65503
  • GENBANK/X65504
  • GENBANK/X66206
  • GENBANK/X66255
  • GENBANK/X71664