ArticleJournal of structural and functional genomics2007
A robust two-step PCR method of template DNA production for high-throughput cell-free protein synthesis.
Article in Journal of structural and functional genomics, 2007. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 30 papers.
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30 citing papers in PubMed, 85 citations in OpenAlex.
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- Amino-acid selective isotope labeling enables simultaneous overlapping signal decomposition and information extraction from NMR spectra.Journal of biomolecular NMR · 2020Article
- Development of aSynthetic biology (Oxford, England) · 2018Article
- Cell-free synthesis of functional antibody fragments to provide a structural basis for antibody-antigen interaction.PloS one · 2018Article
- A reproducible and scalable procedure for preparing bacterial extracts for cell-free protein synthesis.Journal of biochemistry · 2017Article
- Central catalytic domain of BRAP (RNF52) recognizes the types of ubiquitin chains and utilizes oligo-ubiquitin for ubiquitylation.The Biochemical journal · 2017Article
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- A pre-metazoan origin of the CRK gene family and co-opted signaling network.Scientific reports · 2016Article
- Cell-free methods to produce structurally intact mammalian membrane proteins.Scientific reports · 2016Article
- Effect of Glu12-His89 Interaction on Dynamic Structures in HIV-1 p17 Matrix Protein Elucidated by NMR.PloS one · 2016Article
- Direct introduction of neomycin phosphotransferase II protein into apple leaves to confer kanamycin resistance.Plant biotechnology (Tokyo, Japan) · 2016Article
- Content of intrinsic disorder influences the outcome of cell-free protein synthesis.Scientific reports · 2015Article
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Authors and funding
9 authors at 2 institutions in 1 country.
Funding
No grant is acknowledged in the PubMed record.
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.
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