Site-specific 32P-labeling of cytokines, monoclonal antibodies, and other protein substrates for quantitative assays and therapeutic application.
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.
Clark
William A
WA
PBL Biomedical Laboratories, Piscataway, NJ 08854-5900, USA. bclark@pblbio.com
Izotova
Lara
L
Philipova
Doranelly
D
Wu
Wei
W
Lin
Lei
L
Pestka
Sidney
S
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000032:analysis / Q000737:chemistry* / Q000627:therapeutic use; D000937:Antigen-Antibody Reactions; D002417:Cattle; D002648:Child; D016384:Consensus Sequence; D016207:Cytokines / Q000032:analysis / Q000737:chemistry* / Q000627:therapeutic use; D006801:Humans; D018796:Immunoconjugates / Q000032:analysis / Q000737:chemistry* / Q000627:therapeutic use; D007553:Isotope Labeling / Q000379:methods*; D008565:Membrane Proteins; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009369:Neoplasms / Q000532:radiotherapy; D010761:Phosphorus Radioisotopes / Q000032:analysis / Q000737:chemistry* / Q000627:therapeutic use; D010766:Phosphorylation; D025941:Protein Interaction Mapping / Q000379:methods; D011494:Protein Kinases / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D011506:Proteins / Q000032:analysis / Q000737:chemistry / Q000627:therapeutic use; D016499:Radioimmunotherapy; D053633:Receptor, Interferon alpha-beta; D017471:Receptors, Interferon / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000737:chemistry / Q000302:isolation & purification / Q000627:therapeutic use; D013379:Substrate Specificity; D013462:Sulfur Radioisotopes / Q000032:analysis / Q000737:chemistry / Q000627:therapeutic use
D000911:Antibodies, Monoclonal; D016207:Cytokines; D018796:Immunoconjugates; D008565:Membrane Proteins; D010761:Phosphorus Radioisotopes; D011506:Proteins; D017471:Receptors, Interferon; D011993:Recombinant Fusion Proteins; D013462:Sulfur Radioisotopes; D053633:Receptor, Interferon alpha-beta; D011494:Protein Kinases