Reference - Detail
| RRC ID | 51236 |
|---|---|
| Author | Nomoto M, Tada Y. |
| Title | Cloning-free template DNA preparation for cell-free protein synthesis via two-step PCR using versatile primer designs with short 3'-UTR. |
| Journal | Genes Cells |
| Abstract |
Cell-free protein synthesis (CFPS) systems largely retain the endogenous translation machinery of the host organism, making them highly applicable for proteomics analysis of diverse biological processes. However, laborious and time-consuming cloning procedures hinder progress with CFPS systems. Herein, we report the development of a rapid and efficient two-step polymerase chain reaction (PCR) method to prepare linear DNA templates for a wheat germ CFPS system. We developed a novel, effective short 3'-untranslated region (3'-UTR) sequence that facilitates translation. Application of the short 3'-UTR to two-step PCR enabled the generation of various transcription templates from the same plasmid, including fusion proteins with N- or C-terminal tags, and truncated proteins. Our method supports the cloning-free expression of target proteins using an mRNA pool from biological material. The established system is a highly versatile platform for in vitro protein synthesis using wheat germ CFPS. |
| Volume | 23(1) |
| Pages | 46-53 |
| Published | 2018-1-1 |
| DOI | 10.1111/gtc.12547 |
| PMID | 29235215 |
| MeSH | 3' Untranslated Regions* Arabidopsis / genetics Arabidopsis / metabolism Arabidopsis Proteins / metabolism* Cell-Free System* Cloning, Molecular DNA Primers / chemistry DNA Primers / genetics DNA Primers / metabolism DNA, Plant / biosynthesis* DNA, Plant / genetics Polymerase Chain Reaction / methods* Protein Biosynthesis* RNA Processing, Post-Transcriptional* |
| IF | 1.655 |
| Times Cited | 3 |
| Altmetric score |
オルトメトリクス指標項目
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| The most frequently cited source | X(Twitter) |
| Total number of mentions | 3 |
| Altmetric score changes over past 6months | 0.0 |
| Resource | |
| Arabidopsis / Cultured plant cells, genes | pda18109 |