Coding sequences were expressed by coupled transcriptiontranslation in the E. coli cell-free IVTT system. An aliquot of each reaction mixture was analysed by SDS-PAGE and western blot, followed by chemiluminescence of the membrane to visualize the products formed. All four viral (FluHA, FluM, CMVpp65, EBNA3) and one parasite (PyCSP) recombinants could be produced using the manufacturer's recommended conditions. The average yield of each full-length recombinant, detected using an anti-HA antibody directed against the C-Terminal HA tag, was as follow: FluM, 1.15 mg/ml; FluHA, 0.73 mg/ml (two moles of HA tag per molecule of FLU-HA); CMVpp65, 0.7 mg/ml; EBNA3A, 4 ng/ml; and PyCSP, 0.6 mg/ml ( Figures 1A and B and Figure  S1A ). Western blot analysis using an antibody directed against the N-Terminal 6xHIS tag identified the presence of partial products for each viral recombinant (PyCSP not tested) ( Figure S1B ). Solubility analysis showed that a high proportion (at least 70%) of each recombinant was insoluble. A series of optimization studies including kinetics of expression (3 hr to 6 hr), reaction temperature (16uC, 25uC, 30uC), speed (stationary, 300 rpm, 600 rpm), addition of protease inhibitor cocktails, or addition of non-ionic detergents to promote protein solubilisation (Triton X-110 or Figure 1 . Recombinants produced using E. coli cell-free IVTT system. Western Blot and quantification of (A) viral antigens FluM, FluHA, CMVpp65 or EBNA3A and (B) parasite antigen PyCSP pIVEX HisHA IVTT products, probed with mAb against the C-terminal HA tag. Whole IVTT extracts (5 ml) of each antigen were run on a 12% NUPAGE gel, transferred to a PVDF membrane, and probed with anti-HA HRP antibody (1:500 dilution). Protein expression was quantitated against an IVTT-produced recombinant P. falciparum (PF14_0051) protein of known concentration expressing the same N-terminal 6xHis and C-terminal HA tags. Yields for each full length antigen were as follow: FluM, 1.15 mg/ ml; FluHA, 0.73 mg/ ml; CMVpp65, 0.7 mg/ ml; EBNA3A, 4 ng/ ml; and PyCSP, 0.6 mg/ ml. doi:10.1371/journal.pone.0027666.g001 Triton X-114) had no significant effect on the yield of full-length protein or partial products, or protein solubility (data not presented). Accordingly, a standard protocol of 4 hrs incubation at 30uC and 300 rpm was adopted for the IVTT reactions. IVTT products were used either unpurified (whole extract), associated to Polybeads or ProteinG beads, or purified using NI-NTA resin or MagneHis Ni-particles. The MagneHis purification method was associated with a very low recovery yield and this yield was much lower than with the NI-NTA system. For example, for PyCSP, the loss associated with purification using Ni-NTA or MagneHis was approximately 30% and 80% respectively.