*2.1.3 OPH UAA variant expression in cell culture*

Eight OPH mutants containing an amber codon for UAA substitution were generated by GenScript. OPH-H55TAG, H57TAG, H201TAG, H230TAG, H254TAG, H257TAG, D253TAG, and D253E/H254TAG were cloned into pET24b(+) vector using NdeI/XhoI sites for expression in an *E. coli* host. The WT and mutant OPH cloned into pET24b(+) vector were co-transformed into the *E. coli* strain BL21 ai with proper GCE plasmids (**Table 1**). WT-GFP and GFP-150 plasmids were used as positive controls for UAA substitution. The cells were grown in standard LB supplemented with 1 mM UAA and induced by 0.2% L-arabinose and 1 mM IPTG at 16°C overnight. Cells were harvested by centrifugation at 4500 g for 15 min and resuspended in Ni-NTA lysis buffer: 50 mM NaH2PO4, 300 mM NaCl, 10 mM imidazole, and 0.1 μM pepstatin at a pH of 7.4. The cells were lysed using Microfluidizer (Microfluidics Corp, Westwood, MA) and then centrifuged at 12,000 g for 30 min. The collected supernatant was incubated with 5 ml of Ni-NTA resin under end-to-end shaking and loaded on a 5 mL HisTrap FF column. After washing with wash buffer containing 50 mM NaH2PO4, 300 mM NaCl, 20 mM imidazole, the proteins were eluted by elution


#### **Table 1.**

*GCE plasmid and UAA combination used for co-transformation.*

buffer containing 50 mM NaH2PO4, 300 mM NaCl, 250 mM imidazole. Elution fractions containing OPH UAA variants were pooled together and concentrated using a protein concentrator from Thermo Scientific.
