Site-Specific ³² P-Labeling of Cytokines, Monoclonal Antibodies, and Other Protein Substrates for Quantitative Assays and Therapeutic Application

Abstract

Radiolabeled proteins are used in a variety of laboratory applications as well as in radioimmunotherapy. This review focuses on methods that utilize genetic engineering to introduce exogenous phosphorylation sites into proteins. Protein kinase substrate sites can be introduced into target proteins to serve as tags for several purposes. Because many protein kinases, each preferring a unique consensus sequence, are well characterized, the essential structure and function of the target protein can be effectively preserved through judicious selection and design of the phosphate incorporation site. After phosphorylation, these proteins are often indistinguishable from the parent molecules in assays of functional or biological activity. This convenient approach permits incorporation of 32P, 33P, 35S, or nonradioactive 31P, and is rapid, efficient, and safe. Most importantly, 32P labeling of monoclonal antibodies or other therapeutic protein candidates has several significant advantages over radioiodination or chemical conjugation of heavy metal isotopes.

Selenocysteine in proteins—properties and biotechnological use

“…Also here, an affinity tag, is off great use, to specifically localize a radionuclide to a unique site in the recombinant protein. For instance, the introduction of a phosphorylation motif can enable radiolabeling using ATP labeled with phosphor-32 (Chen and Hai, 1994;Clark et al, 2002;Mohanraj et al, 1996) or the use of a His-tag for conjugation of a technetium-99m molecule (Waibel et al, 1999).…”

Site-Specifically Labeled Immunoconjugates for Molecular Imaging—Part 2: Peptide Tags and Unnatural Amino Acids

“…The introduction of this recognition site, ranging from 5 to 15 amino acids depending on the specific kinase used, has been achieved using two strategies: the creation of antibody-based fusion proteins and the genetic engineering of the immunoglobulin. Both of these approaches are discussed in great detail in an excellent review dedicated to the topic [41]. In one example, Patrick et al .…”