*2.1.2 Cell-free protein synthesis of OPH variants*

The rapid cell-free protein expression system can screen hundreds of variants per day in 200 μl reactions. The activity of the expressed product and substrate binding affinity also can be measured directly in the expression mixture, eliminating timeconsuming and labor-intensive cell culture-based protein expression and purification processes. A wheat germ cell-free protein synthesis system from Cell-Free Science was used to generate wild type (WT) and mutant OPH. WT and mutant OPH plasmids were cloned into a pEU-E01 vector. The cell-free expression reaction is detailed here: 20 μl of transcription reactions containing 4 μl of 5x transcription buffer, 2 μl of NTP mix, 0.25 μl of RNase inhibitor, 0.25 μl of SP6 RNA polymerase, and 2 μl of plasmid are prepared and incubated at 37°C for 6 h, and then, translation reactions containing 10 μl of transcription reaction, 0.8 μl of creatine kinase (1 mg/ml), 10 μl of WEPRO, 75 μM of Zn(OAc)2, and 206 μl of SUB-MIX are prepared and incubated at 15°C for 20 h. OPH variant expression in the cell-free reaction mixture was detected using SDS-gel. A series dilution of purified recombinant OPH was used as standards and loaded with cell-free generated OPH to a SDS gel, and OPH variant concentrations were determined by densitometry.

We also explored, for the first time, the possibility of using cell-free protein synthesis to express OPH with UAA substitutions. The small reaction volumes minimize the need for large quantities of UAAs. The plasmid encoding UAA tRNA/tRNA synthetase pair can be co-translated in the same cell-free reaction with expression protein; thus, the expensive and tricky re-engineering of living cellular expression systems is not required to accommodate a new genetic code for UAAs. In addition, the cell-free protein synthesis system's open access to transcription and translation simplifies protein expression optimization. To increase the chance of success, both wheat-germ and PURExpress cell-free system were used in this study. Six OPH mutants containing the amber codon for UAA substitution were constructed into both the PURExpress vector and pEU-E01 vector. Pilots of expression screening were performed with either co-translation of pAcFRS plasmid with OPH mutant plasmids in the same cell-free reaction or substitute reaction with pAcFRs protein and tRNA.

Ten UAAs were screened at various reaction conditions, including 4-acetyl-L-phenylalanine, 3,4 dihydroxy-L-phenylalanine, 1-methyl-L-histine, 4-amino-L-phenylalanine, 4,4,4,4,4,4-hexafluoro-valine, p-fluoro-phenylalanine, 5,5,5-trifluoro-leucine, 3,3,3-trifluoro-alanine, 1–4-thiazolylalanine, and L-3-thienylalanine. Only 4-acetyl-L-phenylalanine showed some level of expression in both cell-free systems, but kinetic measurement did not detect any activity on this UAA substitution. This expression was not confirmed beyond visual observation of a SDS gel band since this substitution was not active kinetically. More work can be done in the future by researchers who desire to advance the cell-free protein expression capability.
