Báo cáo khoa học: Enhancing the protein production levels in Escherichia coli with a strong promoter

Enhancing the protein production levels in Escherichia coli

with a strong promoter

Hanna Tegel, Jenny Ottosson and Sophia Hober

School of Biotechnology, Department of Proteomics, Royal Institute of Technology, AlbaNova University Center, Stockholm, Sweden

Introduction

Recombinant protein production in bacteria represents

a common strategy for obtaining large amounts of a

protein of interest. Although the use of Escherichia coli

has a long tradition in biotechnology, it is still not a

trivial task to determine the optimal production condi￾tions for all proteins. A system that is optimal for the

production of one protein might be nonfunctional for

another. Apart from the conditions of temperature

and induction, the choice of promoter, bacterial strain

and the solubility of the target protein are other

parameters that affect total protein production, as well

as the amount of soluble protein.

Commonly used promoters in E. coli include the T7

promoter, which originates from bacteriophage T7 [1]

and the E. coli lac promoter [2], as well as its modified

form lacUV5 [3]. The synthetic trc promoter [4],

derived from the E. coli trp and lacUV5 promoters, is

also commonly used. The strength of the different

promoters is determined by the relative frequency of

transcription initiation. This is mainly affected by the

affinity of the promoter sequence for RNA polymer￾ase. T7 RNA polymerase is very selective and efficient,

resulting both in a high frequency of transcription ini￾tiation and effective elongation. These features result

Keywords

Escherichia coli; promoter; protein

production; transcription; translation

Correspondence

S. Hober, School of Biotechnology, Division

of Proteomics, Royal Institute of

Technology, AlbaNova University Center,

106 91 Stockholm, Sweden

Fax: +46 8 55378481

Tel: +46 8 55378330

E-mail: [email protected]

(Received 2 July 2010, revised 5 December

2010, accepted 10 December 2010)

doi:10.1111/j.1742-4658.2010.07991.x

In biotechnology, the use of Escherichia coli for recombinant protein pro￾duction has a long tradition, although the optimal production conditions

for certain proteins are still not evident. The most favorable conditions for

protein production vary with the gene product. Temperature and induction

conditions represent parameters that affect total protein production, as well

as the amount of soluble protein. Furthermore, the choice of promoter and

bacterial strain will have large effects on the production of the target pro￾tein. In the present study, the effects of three different promoters (T7, trc

and lacUV5) on E. coli production of target proteins with different charac￾teristics are presented. The total amount of target protein as well as the

amount of soluble protein were analyzed, demonstrating the benefits of

using a strong promoter such as T7. To understand the underlying causes,

transcription levels have been correlated with the total amount of target

protein and protein solubility in vitro has been correlated with the amount

of soluble protein that is produced. In addition, the effects of two different

E. coli strains, BL21(DE3) and Rosetta(DE3), on the expression pattern

were analyzed. It is concluded that the regulation of protein production is

a combination of the transcription and translation efficiencies. Other

important parameters include the nucleotide-sequence itself and the

solubility of the target protein.

Abbreviations

ABP, albumin binding protein; eGFP, enhanced green fluorescent protein; His6, hexahistidyl tag; PrESTs, protein epitope signature tags;

SD, Shine–Dalgarno.

FEBS Journal 278 (2011) 729–739 ª 2011 The Authors Journal compilation ª 2011 FEBS 729