*2.2.2 Cell-free protein synthesis of OPH variants with increased thermal stability and substrate binding affinity*

Despite the high-throughput advantage, a big challenge for many researchers has been how to maintain kinetic activity of expressed protein in cell-free protein synthesis system. Through screening of the cell-free reaction conditions, we have determined that the amount of creatine kinase played a key role in the success of OPH variant expression. Creatine kinase concentration was optimized and a large number of OPH variants were generated successfully using the cell-free protein synthesis system. Kinetic measurements were performed directly using the cell-free reaction mixture. Results of single mutation at D253E and double mutations at D253E/H254R both have improved paraoxon binding. Detailed study on OPH structure and kinetics on these two variants are reported in our recent publication [9]. In a separate study, we were interested in two threonine residues at allosteric location 54 and 199. We have examined the potential to replace threonine with a larger amino acid isoleucine to fill in the gap in OPH structure, therefore, to achieve active site stabilization. Single mutation at T199I and double mutation at T199I/T54I were not able to improve paraoxon binding affinity, but both mutations increased OPH kinetic activity up to 200-fold compared to WT OPH at temperature up to 60°C (manuscript under preparation), which shined light on OP agent remediation that requires OPH to function at elevated temperatures.

### *2.2.3 OPH UAA mutant expression in E. coli host*

UAA substitution at H257 site was selected as an example to show expression level over time. The 4-amino-L-phenylalanine, 3,4-dihydroxy-L-phenylalanine, and

#### **Figure 2.**

*Time course of OPH UAA substitution expression. Three UAAs were tested for expression over a time period of 36 h. GFP plasmids were used as positive control. UAA expression was detected starting at 12 h, and maintained up to 36 h. Letter abbreviations of UAA's full names are colored in red and to label UAA expression on SDS gels.*

3-bromotyrosine substitutions at H257 were successfully expressed (**Figure 2**) over a 36-hour time period. Both 4-amino-L-phenylalanine and 3-bromotyrosine substitutions expressed at a relatively earlier time around 12 h, followed by 3,4-dihydroxy-L-phenylalanine substitution expression starting around 24 h. All substitutions are consistently expressed up to 36 h. The 3-methyl-histine substitution was also successfully expressed at H55, H57, H201, H230, H254, and H257. The 3-methyl-histine mutant expression level was higher than other three UAA substitutions due to close structural similarity to histidine.
