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

Renata E. Stofko-Hahn, Daniel Carr, John D. Scott

Research output: Contribution to journalArticle

85 Citations (Scopus)

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-depandent 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).

Original languageEnglish (US)
Pages (from-to)274-278
Number of pages5
JournalFEBS Letters
Volume302
Issue number3
DOIs
StatePublished - May 18 1992

Fingerprint

Microtubule-Associated Proteins
Calmodulin
Cyclic AMP-Dependent Protein Kinases
Recombinant Proteins
kemptide
Protein Kinases
Purification
Affinity chromatography
Peptides
Phosphorylation
Factor Xa
Egtazic Acid
Affinity Chromatography
Protein Binding
Base Pairing
Sepharose
Fusion reactions
DNA
Proteins
Broncho-Vaxom

Keywords

  • Affinity purification
  • Microtubule associated protein (MAP 2), cAMP-dependent protein kinase (PKA)
  • Recombinant fusion protein

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Molecular Biology

Cite this

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T1 - 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

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AU - Carr, Daniel

AU - Scott, John D.

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AB - 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-depandent 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).

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KW - Recombinant fusion protein

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