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
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Research Support, U.S. Gov't, P.H.S.
MeSH terms
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Amino Acid Sequence
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Base Sequence
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Binding Sites
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Calmodulin / metabolism
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Calmodulin-Binding Proteins / genetics*
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Cyclic AMP / pharmacology*
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DNA / chemistry
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Escherichia coli / genetics
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Factor Xa / metabolism
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Microtubule-Associated Proteins / genetics
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Microtubule-Associated Proteins / isolation & purification*
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Microtubule-Associated Proteins / metabolism
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Molecular Sequence Data
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Oligopeptides / metabolism
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Peptide Fragments / chemistry
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Peptide Fragments / genetics
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Peptide Fragments / isolation & purification*
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Phosphorylation
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Protein Kinases / metabolism*
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Protein Serine-Threonine Kinases*
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Recombinant Fusion Proteins / chemistry
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Recombinant Fusion Proteins / genetics*
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Recombinant Fusion Proteins / isolation & purification*
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Transformation, Bacterial
Substances
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Calmodulin
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Calmodulin-Binding Proteins
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Microtubule-Associated Proteins
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Oligopeptides
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Peptide Fragments
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Recombinant Fusion Proteins
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KFC protein, Gallus gallus
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kemptide
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DNA
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Cyclic AMP
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Protein Kinases
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Protein Serine-Threonine Kinases
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Factor Xa
Associated data
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GENBANK/S86390
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GENBANK/S86392
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GENBANK/S90294
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GENBANK/S90296
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GENBANK/S90298
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GENBANK/X65503
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GENBANK/X65504
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GENBANK/X66206
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GENBANK/X66255
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GENBANK/X71664