Synthesis of lipid-linked oligosaccharides by a compartmentalized multi-enzyme cascade for the in vitro N-glycosylation of peptides
| Autor: | Erdmann Rapp, Valerian Grote, Simon Boecker, Marcus Hoffmann, Udo Reichl, Robert Kottler, Katja Bettenbrock, Anna Schildbach, Markus Pietzsch, Sebastian Tischlik, Christin Bergmann, Thomas Rexer, Reza Mahour, Lisa Wenzel |
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| Rok vydání: | 2020 |
| Předmět: |
Lipopolysaccharides
0106 biological sciences 0301 basic medicine Glycosylation Mannose Bioengineering Disaccharides Endoplasmic Reticulum Nucleotide sugar 01 natural sciences Applied Microbiology and Biotechnology 03 medical and health sciences chemistry.chemical_compound N-linked glycosylation 010608 biotechnology Glycosyltransferase Sf9 Cells Animals chemistry.chemical_classification Cell-Free System biology Endoplasmic reticulum Oligosaccharyltransferase Glycopeptides General Medicine Enzymes 030104 developmental biology chemistry Biochemistry Biocatalysis biology.protein Synthetic Biology Glycoprotein Biotechnology |
| Zdroj: | Journal of Biotechnology. 322:54-65 |
| ISSN: | 0168-1656 |
| Popis: | A wide range of glycoproteins can be recombinantly expressed in aglycosylated forms in bacterial and cell-free production systems. To investigate the effect of glycosylation of these proteins on receptor binding, stability, efficacy as drugs, pharmacodynamics and pharmacokinetics, an efficient glycosylation platform is required. Here, we present a cell-free synthetic platform for the in vitro N-glycosylation of peptides mimicking the endoplasmic reticulum (ER) glycosylation machinery of eukaryotes. The one-pot, two compartment multi-enzyme cascade consisting of eight recombinant enzymes including the three Leloir glycosyltransferases, Alg1, Alg2 and Alg11, expressed in E. coli and S. cerevisiae, respectively, has been engineered to produce the core lipid-linked (LL) oligosaccharide mannopentaose-di-(N-acetylglucosamine) (LL-Man5). Pythanol (C20H42O), a readily available alcohol consisting of regular isoprenoid units, was utilized as the lipid anchor. As part of the cascade, GDP-mannose was de novo produced from the inexpensive substrates ADP, polyphosphate and mannose. To prevent enzyme inhibition, the nucleotide sugar cascade and the glycosyltransferase were segregated into two compartments by a cellulose ester membrane with 3.5 kDa cut-off allowing for the effective diffusion of GDP-mannose across compartments. Finally, as a proof-of-principle, pythanyl-linked Man5 and the single-subunit oligosaccharyltransferase Trypanosoma brucei STT3A expressed in Sf9 insect cells were used to in vitro N-glycosylate a synthetic peptide of ten amino acids bearing the eukaryotic consensus motif N-X-S/T. |
| Databáze: | OpenAIRE |
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