Expression and radiolabeling of recombinant proteins containing a phosphorylation motif.
Radiolabeled proteins are useful in basic and clinical research. Current methods available for radiolabeling proteins involve chemical derivatization, resulting in multiple additions of radionuclides at random sites. A method designed to specifically localize the radionuclide to a unique site will offer advantages of control and predictability in radiolabeling. We have studied the usefulness of a prokaryotic expression vector by incorporating the coding sequence of a consensus phosphorylation motif (Kemptide) for the cAMP-dependent protein kinase A immediately upstream to the multiple cloning site. This vector was used to express five different recombinant proteins with a phosphorylation site at the amino terminus. In addition, the phosphorylation motif was introduced into two other proteins and expressed in yeast. The genetically engineered proteins were purified to homogeneity by affinity chromatography and radiolabeled with [gamma32P]ATP in vitro. All seven proteins used in this study could be expressed with the phosphorylation sequence at their amino terminus and specifically labeled without loss of biological activity. This strategy allows the option of labeling proteins to high or low specific radioactivity and holds potential for in vitro binding and in vivo localization studies.
Mohanraj
D
D
Department of Pharmacology, 3-249 Millard Hall, University of Minnesota, 435 Delaware Street SE, Minneapolis, Minnesota, 55455, USA.
Wahlsten
J L
JL
Ramakrishnan
S
S
D000255:Adenosine Triphosphate / Q000378:metabolism; D001427:Bacterial Toxins; D002455:Cell Division / Q000187:drug effects; D003001:Cloning, Molecular; D016384:Consensus Sequence / Q000235:genetics; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D017931:DNA Primers; D004167:Diphtheria Toxin / Q000378:metabolism / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D016228:Endothelial Growth Factors / Q000378:metabolism; D004768:Enterotoxins / Q000378:metabolism / Q000494:pharmacology; D015870:Gene Expression / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics; D008222:Lymphokines / Q000378:metabolism; D008970:Molecular Weight; D009842:Oligopeptides / Q000235:genetics* / Q000378:metabolism; D010759:Phosphorus Isotopes; D010766:Phosphorylation; D015202:Protein Engineering; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D012276:Ricin / Q000378:metabolism; D018089:Superantigens; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors; D015003:Yeasts / Q000235:genetics
D001427:Bacterial Toxins; D017931:DNA Primers; D004167:Diphtheria Toxin; D016228:Endothelial Growth Factors; D004768:Enterotoxins; D008222:Lymphokines; D009842:Oligopeptides; D010759:Phosphorus Isotopes; D011994:Recombinant Proteins; D018089:Superantigens; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors; C030107:enterotoxin F, Staphylococcal; C037642:kemptide; D000255:Adenosine Triphosphate; D012276:Ricin; D017868:Cyclic AMP-Dependent Protein Kinases