Abstract
Integral membrane proteins have become the focus of interest of many laboratories and structural genomics consortia, but their study is hampered by bottlenecks in production, solubilization, purification and crystallization. In our laboratory we have addressed the problem of high-level protein expression in the membrane of Escherichia coli by use of Mistic, a novel Bacillus subtilis protein, as a fusion partner. In this study we examine the effect of Mistic on protein expression and membrane integration levels of members of the E. coli histidine kinase receptor family. We find that Mistic fusion invariably increases the overall yield by targeting the cargo proteins more efficiently to the membrane and may even replace the signal sequence. Mistic fusion methods will likely be instrumental for high-level expression of other integral membrane proteins.
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Abbreviations
- IMP:
-
Integral membrane protein
- SP:
-
Signal peptide
- HKR:
-
Histidine kinase receptor
- PCR:
-
Polymerase chain reaction
- PBS:
-
Phosphate buffer saline
- M:
-
Mistic fused protein, misticated
- NM:
-
Non-mistic fused, non-misticated
- FL:
-
Full length
- T:
-
Truncated
- MAD:
-
Multi-wavelength anomalous diffraction
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Acknowledgments
We thank Tony Hunter and Jill Meisenhelder (Salk Institute) for providing facilities for, and help with the phosphorylation assays, and Kit Pogliano (UCSD) for providing the E. coli K-12 MG1655 strain. This work is supported by NIH Protein Structure Initiative grant GM074929 and GM74821.
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Kefala, G., Kwiatkowski, W., Esquivies, L. et al. Application of Mistic to improving the expression and membrane integration of histidine kinase receptors from Escherichia coli . J Struct Funct Genomics 8, 167–172 (2007). https://doi.org/10.1007/s10969-007-9033-4
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DOI: https://doi.org/10.1007/s10969-007-9033-4