PMC

(A,B) [³H]-DOPA binding on CHO cells expressing OA1-EGFP. Specific binding was obtained by the subtraction of the binding value with 10 mM DOPA CHE from the total binding. One typical example is shown. (A) Saturation curve. (B) Scatchard plot. The estimated K_D is 81.3 μM from this experiment. (C) Competitive inhibition of 10 μM [³H]-DOPA binding to OA1-EGFP with various concentration of DOPA CHE. Results indicate that DOPA CHE and DOPA compete for the same OA1 binding site. The estimated K_i for DOPA CHE from this experiment is 155.1 μM. (D–G) Representative traces of [Ca²⁺]i during the time course of the standard experimental protocol in ARPE-19 cells transfected with mouse OA1-mCherry. After establishing a stable baseline, the cells were stimulated with 10 μM DOPA. DOPA CHE (10 μM), when used, was applied 10 min before DOPA application. DOPA CHE alone produced no effect. (D,E: upper panels) were inhibited by DOPA CHE pre-incubation (E,F: lower panels). Calcium ionophore (4-Br A23187) was added to confirm the Ca²⁺ imaging (D,F). (G) Summary data for [Ca²⁺]i in response to DOPA and DOPA CHE inhibition. Each bar represents the mean change from baseline [Ca²⁺]i after DOPA stimulation. Data shown are mean ± SEM (n = 5). *P < 0.01, compared with no pre-treatment.

The expression system for the direct incorporation of DOPA biosynthesized from a TPL is shown in . The genes for the evolved aa-tRNA and aaRS pair are placed in a plasmid, and the GFP gene (GFP-E90TAG) containing an amber codon at position 90 is located in another plasmid to evaluate the incorporation of DOPA by GFP fluorescence. The TPL gene is placed in the same expression plasmid containing the GFP gene and constitutively expressed to maximize the yield of the DOPA biosynthesis. Using this expression system, optimal conditions for DOPA biosynthesis were screened. The growth media used for this experiment contained 25 mM ammonia and 100 mM pyruvate, with varying concentrations of catechol (0 - 10 mM). The catechol concentration was critical for the DOPA biosynthesis, while ammonia and pyruvate concentrations over 25 mM and 100 mM, respectively, did not affect the biosynthesis and incorporation of DOPA. The optimal concentration of catechol was 10 mM, and a concentration of more than 10 mM significantly decreased the bacterial cell growth due to its cytotoxicity. In this optimal condition, DOPA was biosynthesized by TPL from its starting materials, and the biosynthesized DOPA was directly incorporated into GFP. The expressed mutant GFP (GFP-E90DOPA) was purified and analyzed by SDS-PAGE (A). The full-length GFP was purified, and the DOPA incorporation was confirmed by MALDI-TOF MS analysis (B). The protein was digested with trypsin, the peptide fragment containing DOPA was analyzed, and the result showed the exclusive incorporation of DOPA with no detectable Tyr or other natural amino acid incorporation. The incorporation efficiency of DOPA biosynthesized by TPL was similar to the incorporation efficiency with 3 mM DOPA, which is the maximum concentration of DOPA because of its cytotoxicity. Although 10 mM catechol showed moderate toxicity, the cell density after 16 h of culture time was three- to fourfold higher than in the presence of 3 mM DOPA. The purified protein yield by biosynthesis was threefold higher than that from the experiment using 3 mM DOPA ().

The expression system for the direct incorporation of DOPA biosynthesized from a TPL is shown in . The genes for the evolved aa-tRNA and aaRS pair are placed in a plasmid, and the GFP gene (GFP-E90TAG) containing an amber codon at position 90 is located in another plasmid to evaluate the incorporation of DOPA by GFP fluorescence. The TPL gene is placed in the same expression plasmid containing the GFP gene and constitutively expressed to maximize the yield of the DOPA biosynthesis. Using this expression system, optimal conditions for DOPA biosynthesis were screened. The growth media used for this experiment contained 25 mM ammonia and 100 mM pyruvate, with varying concentrations of catechol (0 - 10 mM). The catechol concentration was critical for the DOPA biosynthesis, while ammonia and pyruvate concentrations over 25 mM and 100 mM, respectively, did not affect the biosynthesis and incorporation of DOPA. The optimal concentration of catechol was 10 mM, and a concentration of more than 10 mM significantly decreased the bacterial cell growth due to its cytotoxicity. In this optimal condition, DOPA was biosynthesized by TPL from its starting materials, and the biosynthesized DOPA was directly incorporated into GFP. The expressed mutant GFP (GFP-E90DOPA) was purified and analyzed by SDS-PAGE (A). The full-length GFP was purified, and the DOPA incorporation was confirmed by MALDI-TOF MS analysis (B). The protein was digested with trypsin, the peptide fragment containing DOPA was analyzed, and the result showed the exclusive incorporation of DOPA with no detectable Tyr or other natural amino acid incorporation. The incorporation efficiency of DOPA biosynthesized by TPL was similar to the incorporation efficiency with 3 mM DOPA, which is the maximum concentration of DOPA because of its cytotoxicity. Although 10 mM catechol showed moderate toxicity, the cell density after 16 h of culture time was three- to fourfold higher than in the presence of 3 mM DOPA. The purified protein yield by biosynthesis was threefold higher than that from the experiment using 3 mM DOPA ().

The expression system for the direct incorporation of DOPA biosynthesized from a TPL is shown in . The genes for the evolved aa-tRNA and aaRS pair are placed in a plasmid, and the GFP gene (GFP-E90TAG) containing an amber codon at position 90 is located in another plasmid to evaluate the incorporation of DOPA by GFP fluorescence. The TPL gene is placed in the same expression plasmid containing the GFP gene and constitutively expressed to maximize the yield of the DOPA biosynthesis. Using this expression system, optimal conditions for DOPA biosynthesis were screened. The growth media used for this experiment contained 25 mM ammonia and 100 mM pyruvate, with varying concentrations of catechol (0 - 10 mM). The catechol concentration was critical for the DOPA biosynthesis, while ammonia and pyruvate concentrations over 25 mM and 100 mM, respectively, did not affect the biosynthesis and incorporation of DOPA. The optimal concentration of catechol was 10 mM, and a concentration of more than 10 mM significantly decreased the bacterial cell growth due to its cytotoxicity. In this optimal condition, DOPA was biosynthesized by TPL from its starting materials, and the biosynthesized DOPA was directly incorporated into GFP. The expressed mutant GFP (GFP-E90DOPA) was purified and analyzed by SDS-PAGE (A). The full-length GFP was purified, and the DOPA incorporation was confirmed by MALDI-TOF MS analysis (B). The protein was digested with trypsin, the peptide fragment containing DOPA was analyzed, and the result showed the exclusive incorporation of DOPA with no detectable Tyr or other natural amino acid incorporation. The incorporation efficiency of DOPA biosynthesized by TPL was similar to the incorporation efficiency with 3 mM DOPA, which is the maximum concentration of DOPA because of its cytotoxicity. Although 10 mM catechol showed moderate toxicity, the cell density after 16 h of culture time was three- to fourfold higher than in the presence of 3 mM DOPA. The purified protein yield by biosynthesis was threefold higher than that from the experiment using 3 mM DOPA ().