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.
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).
Stofko-Hahn
R E
RE
Vollum Institute, Oregon Health Sciences University, Portland 97201-3098.
Carr
D W
DW
Scott
J D
JD
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D002147:Calmodulin / Q000378:metabolism; D002148:Calmodulin-Binding Proteins / Q000235:genetics*; D000242:Cyclic AMP / Q000494:pharmacology*; D004247:DNA / Q000737:chemistry; D004926:Escherichia coli / Q000235:genetics; D015951:Factor Xa / Q000378:metabolism; D008869:Microtubule-Associated Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification*; D014169:Transformation, Bacterial
D002147:Calmodulin; D002148:Calmodulin-Binding Proteins; D008869:Microtubule-Associated Proteins; D009842:Oligopeptides; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; C074981:KFC protein, Gallus gallus; C037642:kemptide; D004247:DNA; D000242:Cyclic AMP; D011494:Protein Kinases; D017346:Protein Serine-Threonine Kinases; D015951:Factor Xa