ArticleProtein science : a publication of the Protein Society2011
Restrained expression, a method to overproduce toxic membrane proteins by exploiting operator-repressor interactions.
Article in Protein science : a publication of the Protein Society, 2011. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 17 papers.
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Who cites it
17 citing papers in PubMed, 43 citations in OpenAlex.
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- Protein over-expression in Escherichia coli triggers adaptation analogous to antimicrobial resistance.Microbial cell factories · 2021Article
- Structure and function of the juxtamembrane GAF domain of potassium biosensor KdpD.Protein science : a publication of the Protein Society · 2020Article
- Synthetic antibodies against BRIL as universal fiducial marks for single-particle cryoEM structure determination of membrane proteins.Nature communications · 2020Article
- Membrane directed expression in Escherichia coli of BBA57 and other virulence factors from the Lyme disease agent Borrelia burgdorferi.Scientific reports · 2019Article
- Bicistronic Design-Based Continuous and High-Level Membrane Protein Production inACS synthetic biology · 2019Article
- Smoothing membrane protein structure determination by initial upstream stage improvements.Applied microbiology and biotechnology · 2019Review
- Expression of Leptospira membrane proteins Signal Peptidase (SP) and Leptospira Endostatin like A (Len A) in BL-21(DE3) is toxic to the host cells.Journal, genetic engineering & biotechnology · 2018Article
- Biochemical characterization of essential cell division proteins FtsX and FtsE that mediate peptidoglycan hydrolysis by PcsB in Streptococcus pneumoniae.MicrobiologyOpen · 2016Article
- RNA editing by T7 RNA polymerase bypasses InDel mutations causing unexpected phenotypic changes.Nucleic acids research · 2015Article
- In vitro reassembly of the ribose ATP-binding cassette transporter reveals a distinct set of transport complexes.The Journal of biological chemistry · 2015Article
- Design and expression of fusion protein consists of HBsAg and Polyepitope of HCV as an HCV potential vaccine.Advanced biomedical research · 2015Article
- Putative Inv is essential for basolateral invasion of Caco-2 cells and acts synergistically with OmpA to affect in vitro and in vivo virulence of Cronobacter sakazakii ATCC 29544.Infection and immunity · 2014Article
- Biochemical and molecular characterization of barley plastidial ADP-glucose transporter (HvBT1).PloS one · 2014Article
- Identification of the dimer interface of a bacterial Ca(2+)/H(+) antiporter.Biochemistry · 2012Article
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3 authors at 1 institution in 1 country.
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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.
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