Nec279e001mc

Cell-free reactions were prepared through the addition of 37.5% S30 extract [24 (link)], 50% 2x supermix [20 (link)], 3 µg/mL total plasmid, and 2 mM para-azidomethyl-l-phenylalanine (pAMF). M. jannaschii pAMF tRNA synthetase [5 (link)], M. jannaschii amber suppressor tRNA [5 (link)], and T7 RNA polymerase were individually over-expressed in intact E. coli cells and added to the CF reaction as a crude lysate at a final concentration of 1–2% each. For C¹⁴ reactions, 2% v/v C¹⁴-Leucine (Perkin Elmer, NEC279E001MC) was added to each reaction. Reactions of 100 µL were carried out for 16 h at 25 °C in a V-bottom 96-well plate with rotation at 650 rpm in a thermomixer R (Eppendorf). Analysis of the total and soluble IgG titer by C¹⁴ was performed as has been described previously [20 (link)]. Reactions of 1 mL were carried out in flowerplates (M2P Labs, MTP-48-OFF) at 25 °C with rotation at 650 rpm in a thermomixer R (Eppendorf).
Larger reactions were carried out at a 0.25-L scale in a bioreactor (DASgip, Eppendorf, Hamburg, Germany) with temperature, pH, and DO control. pH was maintained at 7.5 for 14 h using 1 M citrate and 1 M KOH. DO was maintained at 20% for 14 h by blending N₂ and O₂ gas. After 14 h, the pH and DO were changed to 8.0 and 80%, respectively, for three hours. IgG titer was determined by A₂₈₀ after purification with PhyNexus Phytip ProPlus columns.

To quantify the suppression of amber codon, the reaction mixture was supplemented with a small amount of ¹⁴C labeled leucine (3 uL per 100 uL reaction, PerkinElmer: NEC279E001MC, 0.1 mCi/mL)^{44 (link)}. The suppression of amber codon at different sites of the heavy or light chain was determined by [¹⁴C]-autoradiograhy of reducing SDS-PAGE gels. Full length trastuzumab heavy chain and suppressed trastuzumab heavy chain variants run at 50 kD on SDS-PAGE; whereas full length light chain and suppressed trastuzumab light chain runs at 25 kD. Non suppressed (truncated) trastuzumab variants run at a lower molecular weight. Amber suppression in the heavy is determined by: $$suppression=\frac{bandintensityofsuppressedheavyorlightchainTAGvariant}{bandintensityofwildtypeheavyorlightchain}$$ Band intensity was determined by ImageQuant (Amersham Biosciences Corp.; Piscataway, NJ).