Isopropyl β d thiogalactoside

Rosetta 2 cells containing pET22b-U2 were obtained in house.[8 (link)] Degenerate primers for site saturation mutagenesis of position 49 were purchased from Integrated DNA Technologies (Skokie, Illinois, USA). Dpn1 enzyme was purchased from New England Biolabs (Ipswich, Massachusetts, USA). Saccharomyces cerevisiae tRNA^Phe, terrific broth media, hexafluoroisopropanol (HFIP), and ammonium acetate solution were obtained from Sigma Aldrich (St. Louis, Missouri, USA). Carbenicillin, chloramphenicol, Isopropyl-β-D-thiogalactoside (IPTG), and lysozyme were purchased from Gold Biotechnology (St. Louis, Missouri, USA). The Ni-NTA His-Bind purification kit and 4–20% SDS gels were purchased from Novagen (Madison, Wisconsin, USA). Hydrochloric acid, 15% TBE-urea gels, Tris-Cl, boric acid, and triethylamine (TEA) were acquired from Fisher Scientific (Waltham, Massachusetts, USA). The synthetic 12-mer oligonucleotide was purchased from Dharmacon (Lafayette, Colorado, USA).

LmFPPS was cloned into pET-28a vector (Novagen) (encoding an N-terminal His tag) and the recombinant plasmid was used to transform Escherichia coli BL21 (DE3) cells ^{28 (link)}. E. coli cells were grown at 37 °C in LB medium (Thermo Fisher Scientific) supplemented with 30 μg/ml kanamycin (GoldBio). Once the OD₆₀₀ reached 0.6-0.8, protein expression was induced by the addition of 0.1 mM isopropyl β-D-thiogalactoside (GoldBio) and the culture was incubated at 37 °C for 3 hours. Cells were harvested by centrifugation at 6000 g for 10 min at 4 °C and stored at −80 °C. LmFPPS mutants, E97F, E97Y, E97W, T164F, T164Y and T164W were generated using the QuikChange Site-Directed Mutagenesis Kit (Stratagene). The forward and reverse primers designed to introduce these mutations are depicted in Table S1. All LmFPPS mutants were expressed in the same way as the WT protein.