Radioactive labeling of recombinant antibody fragments by phosphorylation using human casein kinase II and [gamma-32P]-ATP.
A wide range of antibody fragments can be expressed in bacteria and detected immunochemically via peptide tags. Using specially designed tags, we have developed a strategy for radiolabeling antibody fragments secreted from bacteria. Tagged antibody fragments were secreted either into the bacterial periplasm or the culture medium. The tag was not subject to proteolysis either in the broth or in human plasma. After affinity purification the antibody fragments were phosphorylated with [gamma-32P]ATP and casein kinase II. The labeled fragments were used in a gel band-shift assay to measure antigen binding affinities. In contrast to non site-specific methods such as radioiodination, antibodies labeled with casein kinase II retain full immunoreactivity. Radioactively phosphorylated antibody fragments may have many other applications, including radioimmunoassays and radioimmunotherapy.
Neri
D
D
Cambridge Centre for Protein Engineering-MRC Centre, UK. dario@mrc_lmb.cam.ac.uk
Petrul
H
H
Winter
G
G
Light
Y
Y
Marais
R
R
Britton
K E
KE
Creighton
A M
AM
D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D001483:Base Sequence; D001709:Biotechnology; D047390:Casein Kinase II; D002460:Cell Line; D017931:DNA Primers / Q000235:genetics; D004355:Drug Stability; D006801:Humans; D007128:Immunoglobulin Fragments / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D010761:Phosphorus Radioisotopes; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D018411:Spodoptera
D017931:DNA Primers; D007128:Immunoglobulin Fragments; D010761:Phosphorus Radioisotopes; D011994:Recombinant Proteins; D000255:Adenosine Triphosphate; D047390:Casein Kinase II; D017346:Protein Serine-Threonine Kinases