Abstract
The heterologous expression of membrane proteins driven by T7 RNA polymerase in E. coli is often limited by a mismatch between the transcriptional and translational rates resulting in saturation of the Sec translocon and non-insertion of the membrane protein. In order to optimize the levels of folded, functional inserted protein, it is important to correct this mismatch. In this protocol, we describe the use of titratable strains of E. coli where two small-molecule inducers are used in a bi-variate analysis to optimize the expression levels by fine tuning the transcriptional and translational rates of an eGFP-tagged membrane protein.
Keywords:
Green fluorescent protein (GFP); Membrane protein; RiboTite; Riboswitch; T7 RNA polymerase; T7 lysozyme; Titratable.
MeSH terms
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Animals
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Cloning, Molecular / methods*
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DNA-Directed RNA Polymerases / genetics
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DNA-Directed RNA Polymerases / metabolism
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Escherichia coli / genetics*
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Escherichia coli / growth & development
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Escherichia coli / metabolism
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Gene Expression
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Green Fluorescent Proteins / genetics*
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Green Fluorescent Proteins / metabolism
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Humans
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Membrane Proteins / genetics*
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Membrane Proteins / metabolism
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Protein Biosynthesis
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Recombinant Proteins / genetics
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Recombinant Proteins / metabolism
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Transcription, Genetic
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Transformation, Genetic
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Viral Proteins / genetics
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Viral Proteins / metabolism
Substances
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Membrane Proteins
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Recombinant Proteins
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Viral Proteins
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enhanced green fluorescent protein
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Green Fluorescent Proteins
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bacteriophage T7 RNA polymerase
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DNA-Directed RNA Polymerases