For cell-free protein expression, E. coli b-galactosidase was used as a model antigen. LacZ, encoding E. coli b-galactosidase was cloned into vector pIVEX2.2EM, which allowed T7 promoter-driven expression in prokaryotic cell-free transcription and translation systems. The vector appends a 6-histidine (6-HIS) coding sequence to the C-terminal end of lacZ for efficient purification of the b-galactosidase protein (Amidi et al. 2010) . The E.coli Tuner TM strain, which is devoid of endogenous b-galactosidase (Merck Chemicals Ltd., Nottingham, UK), was used to make S30 bacterial extract as described previously (Amidi et al. 2010) . A coupled in vitro transcription/ translation reaction mixture (further referred to as IVTT mix), consisted of 30% (v/v) S30 extract, 175 lg/mL tRNA, 250 lg/mL creatine kinase, 5.8 mM magnesium acetate, 260U T7 polymerase, and 50% (v/v) lowmolecular-weight mix (LM mix) containing: 110 mM HEPES, 3.4 mM DTT, 2.4 mM ATP, 1.6 mM CTP, 1.6 mM GTP, 1.6 mM UTP, 0.8 M creatine phosphate (CP), 0.65 mM cAMP, 0.05 mM folinic acid, 0.21 M potassium acetate, 27 mM ammonium acetate, 2 mM each of the 20 amino acids, and 8% (v/v) PEG8000, was used for protein synthesis. To initiate protein expression, plasmid DNA was added to the IVTT mix at a final concentration of 20 nM and the reaction mixture was incubated for 3 h at 30°C. Generation of b-galactosidase-producing AnExILs