دورية أكاديمية
Establishing a Cell-Free Glycoprotein Synthesis System for Enzymatic N -GlcNAcylation.
| العنوان: | Establishing a Cell-Free Glycoprotein Synthesis System for Enzymatic N -GlcNAcylation. |
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| المؤلفون: | DeWinter MA; Medical Scientist Training Program, Northwestern University Feinberg School of Medicine, Chicago, Illinois 60611, United States.; Department of Chemical and Biological Engineering, Northwestern University, Evanston, Illinois 60208, United States.; Chemistry of Life Processes Institute, Northwestern University, Evanston, Illinois 60208, United States.; Center for Synthetic Biology, Northwestern University, Evanston, Illinois 60208, United States., Wong DA; Department of Chemical and Biological Engineering, Northwestern University, Evanston, Illinois 60208, United States.; Chemistry of Life Processes Institute, Northwestern University, Evanston, Illinois 60208, United States.; Center for Synthetic Biology, Northwestern University, Evanston, Illinois 60208, United States., Fernandez R; Department of Chemical and Biological Engineering, Northwestern University, Evanston, Illinois 60208, United States.; Chemistry of Life Processes Institute, Northwestern University, Evanston, Illinois 60208, United States.; Center for Synthetic Biology, Northwestern University, Evanston, Illinois 60208, United States., Kightlinger W; Cell-free Protein Synthesis and Microbial Process Development, National Resilience Inc.,, Oakland, California 94606, United States., Thames AH; Medical Scientist Training Program, Northwestern University Feinberg School of Medicine, Chicago, Illinois 60611, United States.; Department of Chemical and Biological Engineering, Northwestern University, Evanston, Illinois 60208, United States.; Chemistry of Life Processes Institute, Northwestern University, Evanston, Illinois 60208, United States.; Center for Synthetic Biology, Northwestern University, Evanston, Illinois 60208, United States.; Division of Allergy and Immunology, Department of Medicine, Northwestern University Feinberg School of Medicine, Chicago, Illinois 60611, United States., DeLisa MP; Robert Frederick Smith School of Chemical and Biomolecular Engineering, Cornell University, Ithaca, New York 14853, United States.; Cornell Institute of Biotechnology, Cornell University, Ithaca, New York 14853, United States., Jewett MC; Department of Bioengineering, Stanford University, Stanford, California 94305, United States. |
| المصدر: | ACS chemical biology [ACS Chem Biol] 2024 Jul 19; Vol. 19 (7), pp. 1570-1582. Date of Electronic Publication: 2024 Jun 27. |
| نوع المنشور: | Journal Article |
| اللغة: | English |
| بيانات الدورية: | Publisher: American Chemical Society Country of Publication: United States NLM ID: 101282906 Publication Model: Print-Electronic Cited Medium: Internet ISSN: 1554-8937 (Electronic) Linking ISSN: 15548929 NLM ISO Abbreviation: ACS Chem Biol Subsets: MEDLINE |
| أسماء مطبوعة: | Original Publication: Washington, D.C. : American Chemical Society, c2006- |
| مواضيع طبية MeSH: | Campylobacter jejuni*/enzymology , Campylobacter jejuni*/genetics , Campylobacter jejuni*/metabolism , Cell-Free System* , Glycoproteins*/metabolism , Glycoproteins*/genetics , Glycoproteins*/chemistry , Acetylglucosamine*/metabolism , Acetylglucosamine*/chemistry , Hexosyltransferases*/metabolism , Hexosyltransferases*/genetics, Glycosylation ; Humans ; Membrane Proteins/metabolism ; Membrane Proteins/genetics ; Bacterial Proteins/metabolism ; Bacterial Proteins/genetics ; Bacterial Proteins/chemistry ; N-Acetylglucosaminyltransferases/metabolism ; N-Acetylglucosaminyltransferases/genetics |
| مستخلص: | N -linked glycosylation plays a key role in the efficacy of many therapeutic proteins. One limitation to the bacterial glycoengineering of human N -linked glycans is the difficulty of installing a single N -acetylglucosamine (GlcNAc), the reducing end sugar of many human-type glycans, onto asparagine in a single step ( N -GlcNAcylation). Here, we develop an in vitro method for N -GlcNAcylating proteins using the oligosaccharyltransferase PglB from Campylobacter jejuni . We use cell-free protein synthesis (CFPS) to test promiscuous PglB variants previously reported in the literature for the ability to produce N -GlcNAc and successfully determine that PglB with an N311V mutation (PglB N311V ) exhibits increased GlcNAc transferase activity relative to the wild-type enzyme. We then improve the transfer efficiency by producing CFPS extracts enriched with PglB N311V and further optimize the reaction conditions, achieving a 98.6 ± 0.5% glycosylation efficiency. We anticipate this method will expand the glycoengineering toolbox for therapeutic research and biomanufacturing. |
| المشرفين على المادة: | 0 (Glycoproteins) V956696549 (Acetylglucosamine) EC 2.4.1.- (Hexosyltransferases) EC 2.4.99.18 (dolichyl-diphosphooligosaccharide - protein glycotransferase) 0 (Membrane Proteins) 0 (Bacterial Proteins) EC 2.4.1.- (N-Acetylglucosaminyltransferases) |
| تواريخ الأحداث: | Date Created: 20240627 Date Completed: 20240719 Latest Revision: 20240719 |
| رمز التحديث: | 20240719 |
| DOI: | 10.1021/acschembio.4c00228 |
| PMID: | 38934647 |
| قاعدة البيانات: | MEDLINE |
Find this issue from the American Chemical Society | تدمد: | 1554-8937 |
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| DOI: | 10.1021/acschembio.4c00228 |