Guidelines for nucleic acid template design for optimal cell-free protein synthesis using an Escherichia coli reconstituted system or a lysate-based system

被引：1

作者：

Asahara, Haruichi ^{[1
]}

Magnelli, Paula ^{[1
]}

Shi, Xiaofeng ^{[1
]}

Tuckey, Corinna ^{[1
]}

Zhou, Ying ^{[1
]}

Samuelson, James C. ^{[1
]}

机构：

[1] New England Biolabs Inc, Ipswich, MA 01938 USA

来源：

RECOMBINANT PROTEIN EXPRESSION: PROKARYOTIC HOSTS AND CELL-FREE SYSTEMS | 2021年 / 659卷

关键词：

TRANSLATION INITIATION; RNA; EXPRESSION; SEQUENCE; GLUCOSE; CODON; GENE;

D O I：

10.1016/bs.mie.2021.07.005

中图分类号：

Q5 [生物化学];

学科分类号：

071010 ; 081704 ;

摘要：

Cell-free protein synthesis is an attractive method for generating enzyme/protein variants for simplified functional analysis as both in vitro protein expression and analysis may often be performed in a single vial or well. Today, researchers may choose from multiple commercial cell lysate products or reconstituted systems which are compatible with either mRNA, linear DNA or plasmid DNA templates. Here we provide guidance for optimal design of the genetic elements within linear and plasmid DNA templates which are required to reliably practice cell-free protein synthesis. Protocols are presented for generating linear DNA templates, and data are presented to show that linear DNA templates may in many cases provide robust protein yields even when employing an Escherichia coli lysate for protein synthesis. Finally, the use of linear DNA templates makes it possible to bypass all cell cultivation steps and proceed from PCR amplification of synthetic DNA to generation of target protein in a matter of hours.

引用

页码：349 / 367

页数：19

共 24 条

[1] Use of signal sequences as an in situ removable sequence element to stimulate protein synthesis in cell-free extracts [J].