Advancements in the Development of HIF-1α-Activated Protein Switches for Use in Enzyme Prodrug Therapy
(A) Sequences of parental switches Haps3 and Haps59. Numbers above indicate the amino acid residues in yCD. Letters below indicate the 1-letter abbreviation amino acid sequence of the corresponding linker. (B) Schematic of the linker libraries created in Haps59. Lines indicate which combinations of N- and C-terminal linkers were combined to comprise the 18 libraries. (C) Schematic of random mutagenesis libraries of Haps59 and Haps3. A * indicates a representative point mutation. (D) Schematic of the construction of libraries of random circular permutations of CH1 that are randomly inserted into the yCD domain of FLAG-tagged yCD. Multiplex PCR was used to create all possible circular permutations of the CH1 domain. Multiplex inverse PCR was used to create all possible insertion sites within yCD. These two populations of DNA were ligated to create the library.