Dihydrofolate reductase, Fusion protein

Dihydrofolate reductase has been used extensively in translocation experiments. A fusion protein with diphtheria toxin A-fragment was shown to be translocated to the cytosol (Klingenberg and Ols-nes, 1996). The translocation was inhibited by methotrexate, which induces a tight folding of the protein. A fusion with a mutated dihydrofolate reductase that does not bind methotrexate tightly was also translocated, and in this case methotrexate was not able to prevent the translocation. This indicates that not only must the toxin A-fragment be unfolded, but the passenger protein must also be able to unfold for translocation to occur. 

FIGURE 3.5 The use of gene fusions (Rosetta Stones) for contextual annotation of hypothetical proteins. The example shows that if an unknown protein and a thymidylate synthase (ThyA) are present in a number of organisms, it is very likely that the unknown protein is a dihydrofolate reductase (DHFR), if its length, sequence, or other physicochemical characteristics are compatible with those expected in a DHFR. 

To successfully perform Y3H screens, the choice of the anchor moiety of the hybrid ligand is important. MTX, as already indicated, shows much promise. It exhibits high affinity (low nanomolar to picomolar) for the monomeric form of E. coli dihydrofolate reductase (eDHFR), which is a small, compact molecule that can be easily expressed as a fusion protein in yeast cells [46], Furthermore, contrary to what is often observed with nonhybrid small molecules, MTX-hybrid molecules appear to generally permeate yeast cells quite readily. At GPC Biotech we have screened over 50 hybrid ligands in which MTX was coupled to various small molecule chemotypes. To date we have not encountered difficulties with cellular uptake of these molecules. Cellular uptake can readily be determined using appropriate competition experiments, as outlined in Fig. 18.2-5. 

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