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

Anoop Narayanan, Marc Ridilla, Dinesh A. Yernool

Research output: Contribution to journalArticle

22 Scopus citations

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 lacoperator site within a T7lac promoter that occur in the absence of the inducer isopropyl b-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.

Original languageEnglish (US)
Pages (from-to)51-61
Number of pages11
JournalProtein Science
Volume20
Issue number1
DOIs
StatePublished - Jan 2011

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology

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