Advancements in the Development of HIF-1α-Activated Protein Switches for Use in Enzyme Prodrug Therapy
(A) Diagram of Ehaps22 and Ehaps08 sequences. Ehaps22 was selected from the linker library containing 2-amino acid N- and C-terminal linkers. Ehaps08 was selected from the random mutagenesis library of Haps59. Numbers above indicate the amino acid residues in yCD. Letters below indicate the 1-letter abbreviation of the amino acid sequence of the corresponding linker or mutation. (B) Representative 5FC dot toxicity assay on cells expressing yCD, Haps59, Ehaps22, or Ehaps08. Serial dilutions of equal density log phase cultures containing either pGA (−HIF-1α, i.e. not expressing HIF-1α) or pGA-HIF (+ HIF-1α, i.e. expressing HIF-1α) were spotted on minimal media plates containing increasing concentrations of 5FC. (C) Quantification of dot toxicity assay. The highest dilution at which growth was observed for each culture on each plate is plotted against the concentration of 5FC in each plate. The number of replicates of each plate is listed above. Error bars represent max and min for N = 2 plates and standard deviation for N = 4. See Fig S1, S2, S3, and S4 for replicate plates.