Optimized protocol for expression and purification of membrane-bound PglB, a bacterial oligosaccharyl transferase
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 cen...
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| Format: | Article |
| Language: | en_US |
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Elsevier
2017
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| Online Access: | http://hdl.handle.net/1721.1/110397 https://orcid.org/0000-0002-5749-7869 |
| _version_ | 1826213539982868480 |
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| author | Jaffee, Marcie Beth Imperiali, Barbara |
| author2 | Massachusetts Institute of Technology. Department of Biology |
| author_facet | Massachusetts Institute of Technology. Department of Biology Jaffee, Marcie Beth Imperiali, Barbara |
| author_sort | Jaffee, Marcie Beth |
| collection | MIT |
| description | 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. |
| first_indexed | 2024-09-23T15:50:54Z |
| format | Article |
| id | mit-1721.1/110397 |
| institution | Massachusetts Institute of Technology |
| language | en_US |
| last_indexed | 2024-09-23T15:50:54Z |
| publishDate | 2017 |
| publisher | Elsevier |
| record_format | dspace |
| spelling | mit-1721.1/1103972022-10-02T04:35:27Z Optimized protocol for expression and purification of membrane-bound PglB, a bacterial oligosaccharyl transferase Jaffee, Marcie Beth Imperiali, Barbara Massachusetts Institute of Technology. Department of Biology Massachusetts Institute of Technology. Department of Chemistry Jaffee, Marcie Beth Imperiali, Barbara 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. National Institutes of Health (U.S.) (Grant GM039334) 2017-06-30T18:34:04Z 2017-06-30T18:34:04Z 2013-06 2013-04 Article http://purl.org/eprint/type/JournalArticle 1046-5928 1096-0279 http://hdl.handle.net/1721.1/110397 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. https://orcid.org/0000-0002-5749-7869 en_US http://dx.doi.org/10.1016/j.pep.2013.04.001 Protein Expression and Purification Creative Commons Attribution-NonCommercial-NoDerivs License http://creativecommons.org/licenses/by-nc-nd/4.0/ application/pdf Elsevier PMC |
| spellingShingle | Jaffee, Marcie Beth Imperiali, Barbara Optimized protocol for expression and purification of membrane-bound PglB, a bacterial oligosaccharyl transferase |
| title | Optimized protocol for expression and purification of membrane-bound PglB, a bacterial oligosaccharyl transferase |
| title_full | Optimized protocol for expression and purification of membrane-bound PglB, a bacterial oligosaccharyl transferase |
| title_fullStr | Optimized protocol for expression and purification of membrane-bound PglB, a bacterial oligosaccharyl transferase |
| title_full_unstemmed | Optimized protocol for expression and purification of membrane-bound PglB, a bacterial oligosaccharyl transferase |
| title_short | Optimized protocol for expression and purification of membrane-bound PglB, a bacterial oligosaccharyl transferase |
| title_sort | optimized protocol for expression and purification of membrane bound pglb a bacterial oligosaccharyl transferase |
| url | http://hdl.handle.net/1721.1/110397 https://orcid.org/0000-0002-5749-7869 |
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