A robust two-step PCR method of template DNA production for high-throughput cell-free protein synthesis
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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 μg/ml, and was used without purification as a template at a concentration of 2–4 μg/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.
KeywordsCell-free protein synthesis Linear template construction Two-step PCR Tagged proteins Human proteins
E. coli maltose binding protein
Streptavidin binding peptide
Tobacco etch virus
The authors thank Dr. Daisuke Kiga (Tokyo Institute of Technology) and Dr. Yasuaki Kawarasaki (University of Shizuoka) for helpful discussions; Dr. Takayoshi Matsuda (RIKEN) for providing HSQC spectra of Ras mutants; Natsuko Matsuda, Natsumi Suzuki, and Yikkiko Fujikura for their technical assistance; and Tomoko Nakayama and Azusa Ishii for expert secretarial assistance. This work was supported by the RIKEN Structural Genomics/Proteomics Initiative (RSGI), the National Project on Protein Structural and Functional Analysis, Ministry of Education, Culture, Sports, Science and Technology of Japan.