Rapid selection of specific MAP kinase-binders from designed ankyrin repeat protein libraries

Abstract

We describe here the rapid selection of specific MAP-kinase binders from a combinatorial library of designed ankyrin repeat proteins (DARPins). A combined in vitro/in vivo selection approach, based on ribosome display and the protein fragment complementation assay (PCA), yielded a large number of different binders that are fully functional in the cellular cytoplasm. Ribosome-display selection pools of four successive selection rounds were examined to monitor the enrichment of JNK2-specific DARPins. Surprisingly, only one round of ribosome display with subsequent PCA selection of this pool was necessary to isolate a first specific binder with micromolar affinity. After only two rounds of ribosome-display selection followed by PCA, virtually all DARPins showed JNK2-specific binding, with affinities in the low nanomolar range. The enrichment factor of ribosome display thus approaches 10(5) per round. In a second set of experiments, similar results were obtained with the kinases JNK1 and p38 as targets. Again, almost all investigated DARPins obtained after two rounds of ribosome display showed specific binding to the targets used, JNK1 or p38. In all three selection experiments the identified DARPins possess very high specificity for the target kinase. Taken together, the combination of ribosome display and PCA selections allowed the identification of large pools of binders at unparalleled speed. Furthermore, DARPins are applicable in intracellular selections and immunoprecipitations from the extract of eukaryotic cells.

ProteomeBinders: planning a European resource of affinity reagents for analysis of the human proteome

“…18 , ribosome display 19,20 , cellsurface display, bacterial two-hybrid, functional colony screening, protein-fragment complementation 21 , SELEX 22 for aptamer selection and combinations of these methods 23 will be compared taking into account the molecular entity being selected and the intended downstream applications (Box 2). For example, if affinity is crucial, technologies with built-in evolution will be required (for example, ribosome display 24 ), whereas if only 'some binder at some epitope' is needed, more technologies become available and selection is less stringent.…”

Structural and functional analysis of phosphorylation-specific binders of the kinase ERK from designed ankyrin repeat protein libraries

“…Affinity precipitation experiments using selected DARPins were performed as described in ref. 19. Endogenous ERK2, pERK, and recombinant DARPins were detected by Western blotting.…”

“…Members of these libraries show very favorable biophysical properties, are expressed in soluble form with high yields in the cytoplasm of Escherichia coli, and do not contain disulfide bonds, allowing for intracellular applications (12,(15)(16)(17)(18). Furthermore, using complex DARPin libraries, specific binders with high affinity and selectivity for their target antigen could be isolated (12,19,20).…”

Affibody molecules: Engineered proteins for therapeutic, diagnostic and biotechnological applications

“…The vast majority of molecular imaging investigations are performed using the glucose analogue 2-[ 18 F]fluoro-2-deoxy-D-glucose ( 18 F-FDG PET), which has proven its value for detection of metastatic and recurrent disease, as well as for detection of early treatment response [23][24][25]. However, 18 F-FDG PET generates a relatively non-specific readout, showing increased glucose metabolism without providing information on the presence of cancerspecific cell-surface receptors such as human epidermal growth factor receptor 2 (HER2), and therefore not providing guidance for receptor-targeted therapy. An ideal imaging agent should have high specificity and affinity for its target.…”

“…18 F is currently the most commonly used positron emitter for PET studies. Magnetic resonance imaging (MRI) is a powerful tool for in vivo anatomical and molecular imaging.…”