title: 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.
authors.affiliation: Protein Research Group, RIKEN Genomic Sciences Center, 1-7-22 Suehiro-cho, Tsurumi, Yokohama 230-0045, Japan.
authors.forename: Takashi
authors.initials: T
authors.lastname: Yabuki
authors.forename: Yoko
authors.initials: Y
authors.lastname: Motoda
authors.forename: Kazuharu
authors.initials: K
authors.lastname: Hanada
authors.forename: Emi
authors.initials: E
authors.lastname: Nunokawa
authors.forename: Miyuki
authors.initials: M
authors.lastname: Saito
authors.forename: Eiko
authors.initials: E
authors.lastname: Seki
authors.forename: Makoto
authors.initials: M
authors.lastname: Inoue
authors.forename: Takanori
authors.initials: T
authors.lastname: Kigawa
authors.forename: Shigeyuki
authors.initials: S
authors.lastname: Yokoyama
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D002474:Cell-Free System; D002478:Cells, Cultured; D004247:DNA / Q000096:biosynthesis*; D017931:DNA Primers / Q000737:chemistry; D004926:Escherichia coli / Q000502:physiology; D006801:Humans; D008969:Molecular Sequence Data; D010957:Plasmids; D016133:Polymerase Chain Reaction / Q000379:methods*; D014176:Protein Biosynthesis; D012689:Sequence Homology, Nucleic Acid
chemical_list: D017931:DNA Primers; D004247:DNA