Direct selection of monoclonal phosphospecific antibodies without prior phosphoamino acid mapping.
In the current post-genomic era, large scale efforts are underway to functionally explore the proteome by assembling large antibody libraries. However, because many proteins are modified post-translationally to regulate their function, collections of modification-specific sensors are also needed. Here we applied a novel approach to select monoclonal phosphospecific antibodies directly from the full-length protein and without up-front phosphoamino acid identification. We chose as antigen GRASP65, a well studied Golgi phosphoprotein. Bacterially produced full-length protein was first incubated with mitotic cytosol, thus allowing modification by naturally occurring kinases, and then used directly for affinity-based antibody selection using a single chain variable fragment phagemid library. In less than 1 week, three distinct and highly functional monoclonal phosphospecific antibodies against two GRASP65 epitopes were obtained and subsequently characterized. The presented approach is carried out fully in vitro, requires no prior knowledge of the phosphoamino acid identity, and is fast and inexpensive. It therefore has great potential to be an attractive alternative to classic animal-based protocols for the selection of post-translation modification sensors and thus to become an invaluable tool in our quest to understand the proteome in all its complexity.
Vielemeyer
Ole
O
Research Section, Translational Department, Institut Curie, 26 Rue d'Ulm, 75248 Paris Cedex 5, France.
Yuan
Hebao
H
Moutel
Sandrine
S
Saint-Fort
Rénette
R
Tang
Danming
D
Nizak
Clément
C
Goud
Bruno
B
Wang
Yanzhuang
Y
Perez
Franck
F
D000818:Animals; D000911:Antibodies, Monoclonal / Q000032:analysis* / Q000276:immunology*; D045163:Antibodies, Phospho-Specific / Q000032:analysis* / Q000276:immunology*; D000915:Antibody Affinity / Q000276:immunology; D000918:Antibody Specificity / Q000276:immunology; D000942:Antigens, Bacterial / Q000276:immunology; D018604:Epitope Mapping; D000077614:Golgi Matrix Proteins; D007135:Immunoglobulin Variable Region / Q000276:immunology; D008565:Membrane Proteins / Q000276:immunology; D018448:Models, Immunological; D019606:Phosphoamino Acids / Q000032:analysis*; D010766:Phosphorylation; D015202:Protein Engineering / Q000379:methods*; D051381:Rats
D000911:Antibodies, Monoclonal; D045163:Antibodies, Phospho-Specific; D000942:Antigens, Bacterial; D000077614:Golgi Matrix Proteins; C491577:Gorasp1 protein, rat; D007135:Immunoglobulin Variable Region; D008565:Membrane Proteins; D019606:Phosphoamino Acids