A custom vector was developed for protein expression in the E. coli cell-free transcription translation system. This vector, called pIVEX HisHA AmpR, was modified from the commercially available pIVEX 2.4d and pIVEX 2.5d vectors (Roche Applied Science, Mannheim, Germany) by incorporating both the N-terminal HIS tag and C-terminal HA tag into the one vector backbone. The HA tag was released from pIVEX 2.5d by digestion with BamHI and XmaI restriction enzymes (New England Biolabs, Ipswich, MA) for 4 hrs at 37uC (1 mg DNA/1 unit enzyme, 20 ml volume) and the fragment then extracted from a 3% agarose gel and purified using a commercially available QIAquick PCR purification kit (Qiagen, Germany) according to manufacturer's instructions. The purified HA-tag was ligated overnight at 16uC into BamHI and XmaI digested pIVEX 2.4d vector using 400 units T4 ligase (New England Biolabs), 25 ng of linearized pIVEX 2.4d vector and 70 ng of the HA-tag insert in a 20 ml reaction. The ligated product was purified from a 1% agarose gel as described above, transformed into TOP10 thermocompetent cells (Invitrogen), and grown on LB plates in the presence of 100 mg/ml ampicillin. Positive colonies were screened by colony PCR using 0.2 units/ml Expand Taq polymerase in Buffer 2 (Roche Diagnostics), 0.4 mM dNTPs and 0.4 mM each primer (forward: TAATACGACTCACTATAGGG; reverse TGCTAGT-TATTGCTCAGCGG) using the following conditions: initial denaturation at 95uC for 5 min; 35 cycles at 95uC for 30 sec, 55uC for 30 sec and 68uC for 1 min; and a final extension at 68uC for 10 min. #NC_007605) and P. yoelii circumsporozoite protein (PyCSP; strain 17XNL, accession #J02695) were amplified by PCR using gene-specific primers flanked with restriction enzyme sites ( Table 1 ). The 50 ml PCR reaction contained 0.2 units/ml Expand Taq polymerase in Buffer 2 (Roche diagnostics), 0.4 mM dNTPs and 0.4 mM each primer and 50 ng DNA template. PCR conditions were: initial denaturation at 95uC for 5 min; 45 cycles at 95uC for 30 sec, 55uC for 30 sec and 68uC for 3 min; and a final extension at 68uC for 10 min. The fragments corresponding to the expected size were excised from a 1% agarose gel, purified using the QIAquick PCR purification kit (Qiagen, Germany), and digested overnight at 37uC using the restrictions enzymes for which the cutting site was present in the flanking sequence. The pIVEX HisHA vector was also digested overnight at 37uC with the same set of restriction enzymes. After cleanup using the PCR purification kit, the gene fragments and the linearized vector were ligated overnight at 16uC at an insert:vector ratio of 1:20 using 30 ng of vector and 400 units T4 ligase (New England Biolabs) in a 20 ml reaction volume. The ligation product was transformed into TOP10 thermo-competent cells, grown overnight at 37uC on LB ampicillin plates (100 mg/ml), and colonies screened by PCR using the same protocol for the customized vector, except that the extension time of the colony PCR was increased to 3 min. One positive colony of each construct was grown overnight at 37uC in LB containing ampicillin (100 mg/ml) and the plasmid purified using the QIAprep Spin Miniprep kit (Qiagen) according to manufacturer's instructions. In a few instances where the IVTT production was poor, the DNA was further purified using phenol/chloroform extraction and ethanol precipitation which improved the yield, but this was not routinely done. All constructs were confirmed by DNA sequencing before use in the IVTT reactions.