Until recently, the principal way to study changes in cellular protein expression levels and perform protein functional analysis was by fluorescent reporter proteins combined with automated confocal imaging systems, both of which provide the basis for high-content screening (HCS) protocols. However, the large size of fluorescent proteins (~26 kD) and relatively high background fluorescence in the cell mandate that the fusion protein be overexpressed for detection, which frequently perturbs the biology being studied.
Immunostaining fixed cells has also been widely used to visualize a protein’s sub-cellular location. However, the time-consuming, laborious labeling procedures are not as amenable to automated fluid handling. Furthermore, high-affinity, selective antibodies suitable for imaging are not always available when a new protein target is identified. These issues are compounded by the fact that HCS protocols generate large amounts of complex data requiring extensive analysis. Although HCS assays have the advantage of multiplexing to simultaneously analyze different cellular events, the infrastructure needed and high instrumentation cost are barriers to routine adoption of HCS in primary screening.
Cell-based high-throughput screening (HTS) assays thus require a technology platform for studying a variety of protein functions in a high-throughput, automated, and flexible manner. DiscoveRx (www.discoverx.com) provides a technology platform of PathHunter assays using a small ProLabel peptide tag. This tag allows the study of many cell functions in live cells, including protein expression, degradation, secretion, translocation, and trafficking, as well as phenotypic events, such as mitosis (Figure 1). Moreover, the flexibility of the ProLabel tag allows screening using one-step homogeneous assay protocols for small molecules or dissecting protein pathways by introducing siRNA into cells. The properties of the ProLabel tag thus afford a flexibility that distinguishes the PathHunter technology as a unique platform with applications in all phases of drug discovery from target identification through lead optimization (Table).
The properties of the ProLabel tag allow high-throughput interrogation of many protein functions in vivo, including quantitating the effects of siRNA in target validation, hit identification, or compound library screening. Biochemically, the ProLabel tag is a small peptide fragment of the well-characterized b-galactosidase (b-gal) enzyme. In the absence of the ProLabel tag, the large b-gal fragment called Enzyme Acceptor (EA) is inactive. However, when the ProLabel peptide combines (complements) with EA, active β-gal is formed.