Griswold Group: Glutathione Transferase Model System
Glutathione transferases serve a variety of functions including detoxification of electrophilic xenobiotics by enzymatically coupling them to the endogenous tripeptide glutathione (GSH). Because of their high degree of structural homology and their widely divergent catalytic activities, these enzymes represent attractive model systems for developing advanced protein engineering tools. Human Glutathione Transferase Theta 1-1 (hGSTT1-1) and rat Glutathione Transferase Theta 2-2 (rGSTT2-2) have been used as model systems to develop novel homology-independent recombination technologies that facilitate the exploration of highly diverse but functional regions of sequence space. Combining these cutting edge library construction techniques with ultra-high throughput flow cytometric screens led to the first experimental isolation of homology-independent chimeras exhibiting catalytic activity equal to or greater than that of wild type enzymes. This work represents a validation of a novel therapeutic enzyme humanization strategy. Motivated by this successful proof of concept, we are now pursuing the development of humanized enzymes with practical therapeutic utility.
Figure 1 – hGSTT1-1 (red) and rGSTT2-2 (blue) are used as parental donors for ITCHY library construction (A) resulting in two complementary single crossover libraries from which RH-A5, RH-F2, HR-216 and HR-25 were isolated; A multiple crossover SCRATCHY library, eSCR-A, is generated using a combination of enhanced crossover SCRATCHY (B1) and RDA-PCR (B2); The library eSCR-A is sorted on a flow cytometer (C) selecting for clones with high CMAC activity; Genes from the selected clones are pooled, combined with the parental hGSTT1-1 gene, and the mixture is subjected to a shuffling step (D) resulting in the humanized library eSCR-B; Library eSCR-B is sorted on a flow cytometer (E) selecting for clones with high CMAC activity; Individual clones are selected for detailed analysis based on whole cell CMAC activity, RFLP analysis and DNA sequencing (F).
Figure 2 – A) Schematic representation of selected chimeric genes. Sequences inherited from hGSTT1-1 are shown as red bars and segments from rGSTT2-2 as blue bars. Insertions not derived from either parent are represented as green bars. Positioning of the progeny segments corresponds to their origin in the parental genes (depicted at top). Point mutations represented as white stars (silent) or green stars (encoding for aa substitution). Sequences encoding the G- and H-sites are noted. B) Mapping of SCR9 aa sequence onto the crystal structure of the hGSTT2 monomer. Human derived sequence in red, rat in blue and identical aa at fusion points in magenta. The location of the active site is denoted with a black star. C) Mapping of SCR23 aa sequence onto the hGSTT2 structure. Point mutations are shown in green.
Figure 3 – Kinetic analysis of selected enzymes' activity. Left: table of Michaelis-Menten parameters for glutathione and 7-amino-4-chloromethyl coumarin (CMAC), the selection substrate used for high throughput screening. Note that several of the engineered enzymes have rate enhancements and catalytic efficiencies exceeding that of the highly active rat parental protein. Right: bar graph of specific activities with CMAC and three alternative substrates. Note that the highly humanized enzyme SCR23 exhibits an entirely orthogonal activity towards ethacrynic acid. This demonstrates the utility of homology-independent recombination for accessing unique regions of functional sequence space.