Tài liệu Báo cáo khoa học: Cell-free translation systems for protein engineering docx

MINIREVIEW

Cell-free translation systems for protein engineering

Yoshihiro Shimizu1

, Yutetsu Kuruma2

, Bei-Wen Ying1

, So Umekage3 and Takuya Ueda1

1 Department of Medical Genome Sciences, Graduate School of Frontier Sciences, The University of Tokyo, Kashiwanoha, Kashiwa-shi,

Chiba, Japan

2 ‘Enrico Fermi’ Center, Compendio del Viminale, Rome, Italy

3 Division of Bioscience and Biotechnology, Department of Ecological Engineering, Toyohashi University of Technology, Tempaku-cho,

Toyohashi, Aichi, Japan

Introduction

Although noncoding RNAs play significant roles in

cellular function [1,2], especially in higher organisms, it

is proteins that dominate most cellular processes. Pro￾teins are the most abundant cellular components and

are responsible for structural, metabolic and regulatory

functions both inside and outside of cells. Thus, inves￾tigation of proteins and elucidation of the molecular

mechanisms underlying their activities are crucial to

our understanding of life.

Generally, owing to their low cost and high produc￾tivity, proteins are prepared using in vivo gene expres￾sion systems. However, the problems associated with

using living cells for recombinant protein expression

include protein degradation and aggregation, or loss of

template DNA. Furthermore, it requires several labori￾ous experimental steps including DNA cloning in the

vector, DNA transformation in cells, and overexpres￾sion of the desired protein in cells. Thus, there are

limitations associated with using in vivo technology for

protein production.

Cell-free translation represents an alternative to

in vivo expression, and rapid progress is being made in

this field, which is gaining attention for its simplicity

and high degree of controllability. Proteins are pro￾duced only when template DNA or mRNA is added

to the reaction mixture, followed by incubation for

Keywords

cell-free protein synthesis; chaperone;

disulfide bond formation; in vitro selection;

liposome; minimal cell; ribosome display;

translation; unnatural amino acid

Correspondence

T. Ueda, Department of Medical Genome

Sciences, Graduate School of Frontier

Sciences, University of Tokyo, FSB401,

5-1-5, Kashiwanoha, Kashiwa-shi, Chiba

prefecture 277-8562, Japan

Fax: +81 4 7136 3642

Tel: +81 4 7136 3641

E-mail: [email protected]

(Received 8 May 2006, revised 20 June

2006, accepted 26 June 2006)

doi:10.1111/j.1742-4658.2006.05431.x

Cell-free translation systems have developed significantly over the last two

decades and improvements in yield have resulted in their use for protein

production in the laboratory. These systems have protein engineering appli￾cations, such as the production of proteins containing unnatural amino

acids and development of proteins exhibiting novel functions. Recently, it

has been suggested that cell-free translation systems might be used as the

fundamental basis for cell-like systems. We review recent progress in the

field of cell-free translation systems and describe their use as tools for pro￾tein production and engineering.

Abbreviations

EGFP, enhanced green fluorescent protein; GFP, green fluorescent protein; PDI, protein disulfide isomerase; PURE, protein synthesis using

recombinant elements; scFv, single-chain variable fragment of antibody; Sec, secretory; SR, signal recognition particle receptor; SRP, signal

recognition particle.

FEBS Journal 273 (2006) 4133–4140 ª 2006 The Authors Journal compilation ª 2006 FEBS 4133