Fusion Dihydrofolate reductase

Successful fusion (2) is a rare event, but the frequency can be improved by adding polyethylene glycol (PEG). To obtain only successfully fused cells, incubation is required for an extended period in a primary culture with HAT medium (3), which contains hypoxan-thine, aminopterin, and thymidine. Amino-pterin, an analogue of dihydrofolic acid, competitively inhibits dihydrofolate reductase and thus inhibits the synthesis of dTMP (see p. 402). As dTMP is essential for DNA synthesis, myeloma cells cannot survive in the presence of aminopterin. Although spleen cells are able to circumvent the inhibitory effect of aminopterin by using hypoxanthine and thymidine, they have a limited lifespan and die. Only hybridomas survive culture in HAT medium, because they possess both the immortality of the myeloma cells and the spleen cells metabolic side pathway. 

Harris JB, Grubb BD, Maltin CA, Dixon R (2000) The neurotoxicity of the venom phospholipases A(2), notexin and taipoxin. Exp Neurol 161 517-26 Haug G, Wilde C, Leemhuis J, Meyer DK, Aktories K et al. (2003) Cellular uptake of Clostridium botulinum C2 toxin membrane translocation of a fusion toxin requires unfolding of its dihydrofolate reductase domain. Biochemistry 42 15284-91 Hauschild A (1993) Epidemiology of human foodborne botulism. In Hauschild A, Dodds KL (eds) Clostridium botulinum ecology and control in foods. Marcel Dekker, Inc. New York, pp 69-104... 

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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