4.2. The Split-Protein Sensor (Split-Trp)

Split-Trp (or protein fragment complementation assay) requires a tryptophan biosynthetic pathway, which is present in mycobacteria. It relies on the reconstitution of an active Trp1p enzyme, only if the POIs interact with each other (Figure 4 and Table 1). This will then allow the tryptophan auxotrophic strain of M. smegmatis ΔhisA to grow on media without tryptophan [83]. The validity of split-Trp was assessed by confirming interactions between ESAT-6 and CFP-10, and the homodimerization of GlfT1 and RegX3 [83]. In parallel with M-PFC, split-Trp was used to evaluate interactions between PknH and DosR. However, only the phosphorylation-defective form of DosR (T198A/T205A) was able to interact with PknH in this system, suggesting that split-Trp is less sensitive than M-PFC [79].

Schematic representation of the split-Trp (or protein fragment complementation assay) technology.