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.

各种糖蛋白可以在细菌和无细胞生产系统中以无糖基化形式重组表达。为了研究这些蛋白质的糖基化对受体结合，稳定性，作为药物的功效，药效学和药代动力学的影响，需要有效的糖基化平台。在这里，我们提出了一种用于模拟真核生物内质网（ER）糖基化机制的肽体外N-糖基化的无细胞合成平台。一锅两室多酶级联反应，由八种重组酶组成，包括三种Leloir糖基转移酶Alg1，Alg2和Alg11，在大肠杆菌和酿酒酵母中表达分别被工程化以产生核心的脂质连接的（LL）寡糖甘露聚糖-二-（N-乙酰基葡糖胺）（LL-Man5）。乙醇（C ₂₀ H ₄₂ O）是一种容易获得的由规则类异戊二烯单元组成的醇，被用作脂质锚。作为级联的一部分，GDP-甘露糖是从廉价的底物ADP，聚磷酸盐和甘露糖重新生产的。为防止酶抑制，核苷酸糖级联反应和糖基转移酶被纤维素酯膜隔离，分隔为两个隔室，截止膜的截留值为3.5 kDa，从而使GDP-甘露糖有效地扩散穿过各个隔室。最后，作为原理的证明，与吡啶基连接的Man5和单亚基寡糖基转移酶在Sf9昆虫细胞中表达的布鲁氏锥虫STT3A用于体外N-糖基化10个带有真核共有基序NXS / T的氨基酸的合成肽。