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Cloning‐free template DNA preparation for cell‐free protein synthesis via two‐step PCR using versatile primer designs with short 3′‐ UTR
Author(s) -
Nomoto Mika,
Tada Yasuomi
Publication year - 2018
Publication title -
genes to cells
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.912
H-Index - 115
eISSN - 1365-2443
pISSN - 1356-9597
DOI - 10.1111/gtc.12547
Subject(s) - cell free protein synthesis , biology , template , computational biology , cloning (programming) , untranslated region , primer (cosmetics) , translation (biology) , protein biosynthesis , microbiology and biotechnology , messenger rna , genetics , gene , computer science , chemistry , organic chemistry , programming language
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 .