Selective in vitro glycosylation of recombinant proteins: semi-synthesis of novel homogeneous glycoforms of human erythropoietin
Selective in vitro glycosylation of recombinant proteins: semi-synthesis of novel homogeneous glycoforms of human erythropoietin
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DOI：
10.1016/s1074-5521(00)90065-6
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发表时间：
2001-02-01
影响因子：
--
通讯作者：
Flitsch, SL
中科院分区：
文献类型：
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作者：
Macmillan, D;Bill, RM;Flitsch, SL
背景：天然糖蛋白通常以一系列糖基化形式存在，其中每个蛋白质分子可能与一系列寡糖结构相关。尽管由于生物样品的微观异质性，人们对结构-功能关系的细节知之甚少，但糖型的整体范围可以具有多种不同的生物物理和生化特性。因此，显然需要能够获得天然和非天然均质糖基化蛋白质的合成方法。已经开发出通过糖基碘乙酰胺与半胱氨酸残基的硫醇基团的选择性反应合成新型糖蛋白，通过定点诱变将其放置在所需的糖基化位点。这提供了合成在天然或非天然糖基化位点携带糖侧链的均质糖基化蛋白质的通用方法。在此，我们证明该方法可应用于糖蛋白激素促红细胞生成素，这是一种重要的治疗性糖蛋白，具有三个 N-糖基化位点，这对于体内生物活性至关重要。 结果：野生型重组促红细胞生成素和三个突变体，其中糖基化位点天冬酰胺残基已更改为半胱氨酸（His(10)-WThEPO、His(10)-Asn24Cys、 His(10)-Asn38Cys、His(10)-Asn83CyshEPO)在大肠杆菌中过表达并纯化，产量为13 mg l(-1)。糖基-β-N-碘乙酰胺的化学糖基化可以通过电喷雾质谱监测。在野生型和突变蛋白中，没有观察到参与二硫键的其他四个半胱氨酸残基的潜在副反应。糖基化的产率通常约为50%，并且使用凝集素亲和层析可以容易地从非糖基化蛋白质中纯化糖基化蛋白质。对纯化糖蛋白的动态光散射分析表明，产生的糖形式是单体，并且与野生型蛋白折叠相同。 结论：即使在存在两个二硫键的情况下，在天然糖基化位点带有特定 Asn drop Cys 突变的大肠杆菌中表达的促红细胞生成素也可以使用 β-N-糖基碘乙酰胺进行糖基化。这些发现为进一步阐述聚糖结构和开发这种合成半合成糖蛋白的通用方法奠定了基础。 (C) 2001 Elsevier Science Ltd. 保留所有权利。
Background: A natural glycoprotein usually exists as a spectrum of glycosylated forms, where each protein molecule may be associated with an array of oligosaccharide structures. The overall range of glycoforms can have a variety of different biophysical and biochemical properties, although details of structure-function relationships are poorly understood, because of the microheterogeneity of biological samples. Hence, there is clearly a need for synthetic methods that give access to natural and unnatural homogeneously glycosylated proteins. The synthesis of novel glycoproteins through the selective reaction of glycosyl iodoacetamides with the thiol groups of cysteine residues, placed by site-directed mutagenesis at desired glycosylation sites has been developed. This provides a general method for the synthesis of homogeneously glycosylated proteins that carry saccharide side chains at natural or unnatural glycosylation sites. Here, we have shown that the approach can be applied to the glycoprotein hormone erythropoietin, an important therapeutic glycoprotein with three sites of N-glycosylation that are essential for in vivo biological activity.Results: Wild-type recombinant erythropoietin and three mutants in which glycosylation site asparagine residues had been changed to cysteines (His(10)-WThEPO, His(10)-Asn24Cys, His(10)-Asn38Cys, His(10)-Asn83CyshEPO) were overexpressed and purified in yields of 13 mg l(-1) from Escherichia coli. Chemical glycosylation with glycosyl-beta -N-iodoacetamides could be monitored by electrospray MS. Both in the wild-type and in the mutant proteins, the potential side reaction of the other four cysteine residues tall involved in disulfide bonds) were not observed. Yield of glycosylation was generally about 50% and purification of glycosylated protein from non-glycosylated protein was readily carried out using lectin affinity chromatography. Dynamic light scattering analysis of the purified glycoproteins suggested that the glycoforms produced were monomeric and folded identically to the wild-type protein.Conclusions: Erythropoietin expressed in E. coli bearing specific Asn drop Cys mutations at natural glycosylation sites can be glycosylated using beta -N-glycosyl iodoacetamides even in the presence of two disulfide bonds. The findings provide the basis for further elaboration of the glycan structures and development of this general methodology for the synthesis of semi-synthetic glycoproteins. (C) 2001 Elsevier Science Ltd. All rights reserved.