Optimized protocol for expression and purification of membrane-bound PglB, a bacterial oligosaccharyl transferase
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Imperiali_Optimized protocol.pdf
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1.59 MB
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Adobe PDF
Checksum (MD5)
a3b7d97314c559d4cb07dcf19100bb4e
Author(s)
Jaffee, Marcie Beth
Imperiali, Barbara
Date Issued
June 2013
Journal
Protein Expression and Purification
Publisher
Elsevier
Citation
Jaffee, Marcie B., and Barbara Imperiali. “Optimized Protocol for Expression and Purification of Membrane-Bound PglB, a Bacterial Oligosaccharyl Transferase.” Protein Expression and Purification 89.2 (2013): 241–250.
Version
Author's final manuscript
Abstract
Asparagine-linked glycosylation (NLG) plays a significant role in a diverse range of cellular processes, including protein signaling and trafficking, the immunologic response, and immune system evasion by pathogens. A major impediment to NLG-related research is an incomplete understanding of the central enzyme in the biosynthetic pathway, the oligosaccharyl transferase (OTase). Characterization of the OTase is critical for developing ways to inhibit, engineer, and otherwise manipulate the enzyme for research and therapeutic purposes. The minimal understanding of this enzyme can be attributed to its complex, transmembrane structure, and the resulting instability and resistance to overexpression and purification. The following article describes an optimized procedure for recombinant expression and purification of PglB, a bacterial OTase, in a stably active form. The conditions screened at each step, the order of screening, and the method of comparing conditions are described. Ultimately, the following approach increased expression levels from tens of micrograms to several milligrams of active protein per liter of Escherichia coli culture, and increased stability from several hours to greater than six months post-purification. This represents the first detailed procedure for attaining a pure, active, and stable OTase in milligram quantities. In addition to presenting an optimized protocol for expression and purification of PglB, these results present a general guide for the systematic optimization of the expression, purification, and stability of a large, transmembrane protein.
MIT Department
Massachusetts Institute of Technology. Department of Biology
Massachusetts Institute of Technology. Department of Chemistry
Terms of Use
Creative Commons Attribution-NonCommercial-NoDerivs License
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DOI of Published Version
https://doi.org/10.1016/j.pep.2013.04.001
