Forceful large-scale expression of “problematic” membrane proteins
We developed an Escherichia coli expression system for overproduction of a highly toxic membrane protein that is impossible to overexpress by traditionally used approaches. The method is based on combination of the genetic modifications of a bicistronic expression plasmid, stabilization of a synthes...
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Published in | Biochemical and biophysical research communications Vol. 327; no. 3; pp. 650 - 655 |
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Main Authors | , , , |
Format | Journal Article |
Language | English |
Published |
United States
Elsevier Inc
18.02.2005
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Subjects | |
Online Access | Get full text |
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Summary: | We developed an
Escherichia coli expression system for overproduction of a highly toxic membrane protein that is impossible to overexpress by traditionally used approaches. The method is based on combination of the genetic modifications of a bicistronic expression plasmid, stabilization of a synthesized protein, and selection of a compatible expression host. This enabled us to enhance the expression level of a toxic membrane protein 30–50 times compared with expression in the native state and to obtain 3–5
mg of a highly purified functionally active protein per liter of culture. We describe the method for the amplified expression of membrane proteins, using the
Pseudomonas aeruginosa multidrug resistance protein, MexY, as an example. The amplified MexY was correctly folded in the cytoplasmic membrane of the
E. coli without forming inclusion bodies. This method can be applicable to the large-scale expression of the other problematic membrane proteins that are otherwise extremely difficult to overproduce. |
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Bibliography: | ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23 |
ISSN: | 0006-291X 1090-2104 |
DOI: | 10.1016/j.bbrc.2004.12.059 |