A robust two-step PCR method of template DNA production for high-throughput cell-free protein synthesis

Abstract

A two-step PCR method has been developed for the robust, high-throughput production of linear templates ready for cell-free protein synthesis. The construct made from the cDNA expresses a target protein region with N- and/or C-terminal tags. The procedure consists only of mixing, dilution, and PCR steps, and is free from cloning and purification steps. In the first step of the two-step PCR, a target region within the coding sequence is amplified using two gene-specific forward and reverse primers, which contain the linker sequences and the terminal sequences of the target region. The second PCR concatenates the first PCR product with the N- and C-terminal double-stranded fragments, which contain the linker sequences as well as the sequences for the tag(s) and the initiation and termination, respectively, for T7 transcription and ribosomal translation, and amplifies it with the universal primer. Proteins can be fused with a variety of tags, such as natural poly-histidine, glutathione-S-transferase, maltose-binding protein, and/or streptavidin-binding peptide. The two-step PCR method was successfully applied to 42 human target protein regions with various GC contents (38-77%). The robustness of the two-step PCR method against possible fluctuations of experimental conditions in practical use was explored. The second PCR product was obtained at 60-120 microg/ml, and was used without purification as a template at a concentration of 2-4 microg/ml in an Escherichia coli coupled transcription-translation system. This combination of two-step PCR with cell-free protein synthesis is suitable for the rapid production of proteins in milligram quantities for genome-scale studies.

RING Domain Mutations Uncouple TRIM5α Restriction of HIV-1 from Inhibition of Reverse Transcription and Acceleration of Uncoating

“…The DNA fragments encoding the wild-type or mutant RING domain of TRIM5␣ rh (1 to 79) and TRIM5␣ hu (1 to 78) were amplified via PCR as a fusion with an N-terminal His tag and tobacco etch virus (TEV) protease cleavage site (56). The 15 N-labeled proteins were produced by cell-free protein synthesis with optimization for zinc-binding proteins (25,33) and were purified by immobilized metal affinity chromatography using an automated system (2).…”

Direct introduction of neomycin phosphotransferase II protein into apple leaves to confer kanamycin resistance

“…The His-tag used in this study was a modified version of the natural poly-histidine tag (Yabuki et al 2007). A dialysis-mode cell-free protein synthesis method, which was previously reported (Spirin et al 1988), was used to synthesize NPTII protein.…”

“…The amplified DNA fragment was cloned into the NcoI and BamHI sites of pET28b(+), and the sequence was confirmed. The NPTII gene cloned was then amplified for cell-free synthesis by a two-step PCR, according to previous reports (Yabuki et al 2007). Briefly, the first PCR was carried out in a 20 µl reaction mixture with 3 µl of a 50-fold diluted buffer, 50 nM each of forward (FW) and reverse (RV) unique primers for NPTII, 0.2 mM of each deoxyribonucleotide triphosphate (dNTP), 1×Expand Hi-Fi buffer (Roche, Basel, Switzerland), and 0.5 U Expand Hi-Fi enzyme (Roche) with a hot start.…”

Production of functional bacteriorhodopsin by an Escherichia coli cell‐free protein synthesis system supplemented with steroid detergent and lipid

“…23 The presence of the N-terminal modification with the Natural polyhistidine (NHis) tag effectively increased the expression yields in a cell-free expression system. 23 BR has a hydrophobic 13 amino acid leader sequence at the N-terminus, which might become unstable in the lipid environment or influence the insertion into the membrane. 24 BR is not processed in the E. coli expression systems.…”

“…23 The second PCR was carried out using the first PCR product, the T7P-NHis-TEV-NL1 and T7T-DT2 fragments, and the U2 universal primer. 23 The T7P-NHis-TEV-NL1 fragment contained the sequences of the T7 promoter, the NHis (Natural polyhistidine) tag, and the TEV (Tobacco Etch Virus) protease site. The NHis tag is a modified version of the HAT tag.…”