Linking binders to applications
Procedures and binding reagents must be appropriate to foreseen applications. Thus, for protein quantification, binders will be produced that either recognise native proteins (e.g. for immunoprecipitation) and/or their denatured forms (e.g. for Western blots), while paired binding reagents against non-overlapping epitopes of the same target, together with a novel RNAi cell array platform, will facilitate validation of specificity and provide convincing controls. To achieve the appropriate binder properties for diverse applications requires careful antigen design, from fully folded proteins or individual domains to unique peptide sequences and (sequence specific) PTMs. In some cases, binders will be derivatised by addition of detection and amplification moieties. An example of the latter is the ultrasensitive proximity ligation method where binders are conjugated with oligonucleotides for subsequent detection by rolling circle amplification (see below). In vivo autobiotinylation of scFv or the addition of Fc domains from different species, e.g. for the use of more than one binder in confocal colocalization sudies, are other options.
One of the key requirements is to have reagents, not only for target detection per se but also for purification and functional testing, notably inside the cell. Intracellular acting binders, aka intrabodies, are poised to fulfil their long-awaited potential as high throughput investigative tools for pathway and interaction analysis and functional disruption. Their varied mode of action gives them great potential in functional proteomics for characterisation of novel gene products and subsequent validation as potential drug targets, and may ultimately become therapeutic entities in their own right. The availability of comprehensive sets of reliable reagents will also underpin progress in intracellular systems biology. Intrabodies have been successfully applied, initially in the scFv format, by UZH and TUBS, to inhibit the function of target proteins in specific cellular compartments. However, a limitation of scFv fragments as intracellular reagents is that, due to the absence of disulphide bond formation in the cytoplasm, their stability is reduced, such that only a subset will function intracellularly. Therefore, there is a great advantage in using a framework such as the DARPin or Affibody scaffold where all members can fold in the absence of disulphide bonds and which do not have unpaired cysteines. Nanobodies also contain the conserved immunoglobulin domain, however, it has been shown that their intracellular expression under no circumstances impedes the antigen binding capacity. Probably, the folding in soluble entities and the intrinsic stability of the nanobodies is sufficiently high to tolerate the absence of the disulphide bond formation.
The intracellular action of binding proteins invites comparison with inhibitory RNA. While RNAi has been widely used to test the importance of particular proteins by inhibiting their synthesis, and its generation is comparatively easy, as only the gene sequence is needed, there are several limitations that can be overcome by a protein approach. (i) The effect of RNAi knock down is often not very strong, especially for long-lived proteins, since only the de novo synthesis of the protein is (partially) inhibited; (ii) with RNAi it is not possible to dissect the effect to domains or modifications; (iii) off-target effects of RNAi have been reported in the literature; and most importantly, (iv) RNA based inhibitors do not allow any protein based validation experiments, whereas a protein-reactive binder can be used directly for proteomics assays including pull-downs, Western Blots, enrichment for MS characterisation, and even co-crystallisation. It must be stressed that RNAi experiments will thus always have to be complemented by experiments using protein binders; in contrast, it is possible that (certain) protein binders can also take on the functional (inhibitory) role of RNAi.
The exciting use of binders as intracellular reagents will fuel the discovery of function of the many proteins whose intracellular actions remain poorly understood or unknown. Intrabodies have yet to reach their potential in clinical transfer and commercial exploitation and further exemplification in this project could greatly improve their development in these areas.