Establishing a Cell-Free Glycoprotein Synthesis System for Enzymatic N-GlcNAcylation.
ACS Chem Biol
; 19(7): 1570-1582, 2024 Jul 19.
Article
em En
| MEDLINE
| ID: mdl-38934647
ABSTRACT
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 (PglBN311V) exhibits increased GlcNAc transferase activity relative to the wild-type enzyme. We then improve the transfer efficiency by producing CFPS extracts enriched with PglBN311V 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.
Texto completo:
1
Base de dados:
MEDLINE
Assunto principal:
Acetilglucosamina
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Glicoproteínas
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Sistema Livre de Células
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Campylobacter jejuni
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Hexosiltransferases
Limite:
Humans
Idioma:
En
Ano de publicação:
2024
Tipo de documento:
Article