Cell-free synthesis of 059-152-Fv and Fab

The dialysis mode of cell-free synthesis was used to synthesize 059-152-Fv and Fab. A small-scale dialysis unit (30 μl internal and 300 μl external solution) was used to optimize the reaction conditions [11]. The composition of the internal solution was 60 mM HEPES-KOH buffer (pH 7.5) containing 200 mM potassium L-glutamate, 15 mM magnesium acetate, 1.5 mM each amino acid, 1.3 mM ATP, 0.9 mM GTP, 0.9 mM CTP, 0.9 mM UTP, 81 μM folinic acid, 27.6 mM ammonium acetate, 80 mM creatine phosphate, 1 mM reduced glutathione (GSH), 4 mM oxidized glutathione (GSSG), 175 μg/ml tRNAs, 0.2 to 0.8 mg/ml disulfide isomerase (DsbC), 100 μg/ml creatine kinase, 60 μg/ml T7 RNA polymerase, 30% (v/v) E. coli S30 extract, and 2 μg/ml each template DNA. To produce the Fv, pCR2.1-059-152-VH and pCR2.1-059-152-VL were added to the internal solution. The plasmids pCR2.1-059-152-VHCH1 and pCR2.1-059-152-LC were added to produce the Fab. Variable concentrations of E. coli DsbC were added to the internal solution to determine its optimal concentration. The external solution was composed of the same components as the internal solution except that DsbC, creatine kinase, T7 RNA polymerase, and template DNAs were omitted. In addition, dithiothreitol-free S30 buffer was used instead of S30 extract [26]. The cell-free reaction was performed at 25°C for 7 h, unless otherwise noted.