Physiological response to membrane protein overexpression in E. coli
| Autor: | Nathan K. Karpowich, Francesca Gubellini, Grégory Verdon, Grégory Boël, Jon D. Luff, Samuel K. Handelman, John F. Hunt, Sarah E. Ades, Nils C. Gauthier |
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| Přispěvatelé: | Columbia University [New York], Pennsylvania State University (Penn State), Penn State System, This work was supported by National Institutes of Health grants 1R21GM07595933 and 1R01GM072867 to J. F. Hunt, National Institutes of Health grants 1U54GM75026 and 5U54GM075026 to W. A. Hendrickson, and National Science Foundation grant MCB-0347302 to S. E. Ades. |
| Rok vydání: | 2011 |
| Předmět: |
Protein Folding
Transcription Genetic MESH: Protein Folding [PHYS.PHYS.PHYS-BIO-PH]Physics [physics]/Physics [physics]/Biological Physics [physics.bio-ph] Population Genetic Vectors Protein Array Analysis MESH: Escherichia coli Proteins [SDV.BC]Life Sciences [q-bio]/Cellular Biology Biology Biochemistry Analytical Chemistry 03 medical and health sciences MESH: Genetic Vectors [CHIM.CRIS]Chemical Sciences/Cristallography Escherichia coli [SDV.BBM]Life Sciences [q-bio]/Biochemistry Molecular Biology RNA Messenger education Molecular Biology Integral membrane protein 030304 developmental biology MESH: RNA Messenger Genetics Regulation of gene expression 0303 health sciences education.field_of_study MESH: Gene Expression Regulation Bacterial 030306 microbiology MESH: Escherichia coli MESH: Protein Array Analysis MESH: Transcription Genetic Escherichia coli Proteins Research Membrane Proteins MESH: Transcription Factors Gene Expression Regulation Bacterial Cell biology [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry Molecular Biology/Biomolecules [q-bio.BM] Membrane protein Membrane biogenesis bacteria Protein folding Target protein MESH: Membrane Proteins [INFO.INFO-BI]Computer Science [cs]/Bioinformatics [q-bio.QM] Biogenesis Transcription Factors |
| Zdroj: | Molecular & Cellular Proteomics : MCP Molecular and Cellular Proteomics Molecular and Cellular Proteomics, American Society for Biochemistry and Molecular Biology, 2011, 10 (10), pp.M111.007930. ⟨10.1074/mcp.M111.007930⟩ |
| ISSN: | 1535-9484 1535-9476 |
| DOI: | 10.1074/mcp.M111.007930⟩ |
| Popis: | International audience; Overexpression represents a principal bottleneck in structural and functional studies of integral membrane proteins (IMPs). Although E. coli remains the leading organism for convenient and economical protein overexpression, many IMPs exhibit toxicity on induction in this host and give low yields of properly folded protein. Different mechanisms related to membrane biogenesis and IMP folding have been proposed to contribute to these problems, but there is limited understanding of the physical and physiological constraints on IMP overexpression and folding in vivo. Therefore, we used a variety of genetic, genomic, and microscopy techniques to characterize the physiological responses of Escherichia coli MG1655 cells to overexpression of a set of soluble proteins and IMPs, including constructs exhibiting different levels of toxicity and producing different levels of properly folded versus misfolded product on induction. Genetic marker studies coupled with transcriptomic results indicate only minor perturbations in many of the physiological systems implicated in previous studies of IMP biogenesis. Overexpression of either IMPs or soluble proteins tends to block execution of the standard stationary-phase transcriptional program, although these effects are consistently stronger for the IMPs included in our study. However, these perturbations are not an impediment to successful protein overexpression. We present evidence that, at least for the target proteins included in our study, there is no inherent obstacle to IMP overexpression in E. coli at moderate levels suitable for structural studies and that the biochemical and conformational properties of the proteins themselves are the major obstacles to success. Toxicity associated with target protein activity produces selective pressure leading to preferential growth of cells harboring expression-reducing and inactivating mutations, which can produce chemical heterogeneity in the target protein population, potentially contributing to the difficulties encountered in IMP crystallization. |
| Databáze: | OpenAIRE |
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