Format

Send to

Choose Destination
Protein Sci. 2011 Jan;20(1):51-61. doi: 10.1002/pro.535.

Restrained expression, a method to overproduce toxic membrane proteins by exploiting operator-repressor interactions.

Author information

1
Department of Biological Sciences, Purdue University, West Lafayette, Indiana 47907, USA.

Abstract

A major rate-limiting step in determining structures of membrane proteins is heterologous protein production. Toxicity often associated with rapid overexpression results in reduced biomass along with low yields of target protein. Mitigation of toxic effects was achieved using a method we call "restrained expression," a controlled reduction in the frequency of transcription initiation by exploiting the infrequent transitions of Lac repressor to a free state from its complex with the lac-operator site within a T7lac promoter that occur in the absence of the inducer isopropyl β-D-1-thiogalactopyranoside. In addition, production of the T7 RNA polymerase that drives transcription of the target is limited using the tightly regulated arabinose promoter in Escherichia coli strain BL21-AI. Using this approach, we can achieve a 200-fold range of green fluorescent protein expression levels. Application to members of a family of ion pumps results in 5- to 25-fold increases in expression over the benchmark BL21(DE3) host strain. A viral ion channel highly toxic to E. coli can also be overexpressed. In comparative analyses, restrained expression outperforms commonly used E. coli expression strategies. The mechanism underlying improved target protein yield arises from minimization of protein aggregation and proteolysis that reduce membrane integrity and cell viability. This study establishes a method to overexpress toxic proteins.

PMID:
21031485
PMCID:
PMC3047061
DOI:
10.1002/pro.535
[Indexed for MEDLINE]
Free PMC Article

Supplemental Content

Full text links

Icon for Wiley Icon for PubMed Central
Loading ...
Support Center