School of Health and Life Sciences, Teesside University, Middlesbrough, TS1 3BA, UK; National Horizons Centre, Teesside University, 38 John Dixon Ln, Darlington, DL1 1HG, UK; Glycoselect Ltd., South Tees Institute for Learning, Research and Innovation, the James Cook University Hospital, Middlesbrough, TS4 3BW, UK.
Glycoselect Ltd., South Tees Institute for Learning, Research and Innovation, the James Cook University Hospital, Middlesbrough, TS4 3BW, UK.
Anal Chim Acta. 2021 Apr 22;1155:338352. doi: 10.1016/j.aca.2021.338352. Epub 2021 Feb 27.
Development of biosimilars is costly, where glycan analysis is a significant constraint on time and money. This paper provides an in-depth characterisation of several novel recombinant prokaryotic lectins (RPLs), developed through directed evolution, displaying specific binding activities to α-mannose, β-galactose, fucose and sialic acid residues, tested against major biosimilar targets. The binding characterisation of all lectins was performed employing the principles of bio-layer interferometry (BLI), with help of the streptavidin-coated sensor with the biotinylated lectins. The binding activity of the RPLs and the specificity to a broad range of glycoproteins and glycoconjugates were evaluated and compared to those of equivalent plant-derived lectins. While exhibiting better or similar specificity, RPLs displayed significantly better binding in all cases. The binding mechanisms are explained with particular focus on the role hydrogen bonding plays in the change of specificity for a galactose specific lectin. Furthermore, different sets of RPLs and their plant equivalents were assayed against the different glycoprotein targets to evaluate the analytical parameters of the lectin-glycoprotein interaction. The obtained LoDs reached by the RPLs were lower than those of their plant counterparts apart from one, exhibiting RPL:PL LoD ratios of 0.8, 2.5, 14.2 and 380 for the sets of lectins specific to fucose, α-mannose, β-galactose and sialic acid, respectively. Such enhancement in analytical parameters of RPLs shows their applicability in protein purification and as bioanalytical tools for glycan analysis and biosensor development.
生物类似药的开发成本高昂，其中聚糖分析在时间和金钱上是一个重大的限制。本文深入描述了几种通过定向进化开发的新型重组原核凝集素（RPL），这些凝集素具有特定的结合活性，可以结合α-甘露糖、β-半乳糖、岩藻糖和唾液酸残基，针对主要的生物类似药靶标进行了测试。所有凝集素的结合特性都是通过生物层干涉（BLI）原理来进行的，使用链霉亲和素包被的传感器和生物素化的凝集素来进行。评估了 RPL 的结合活性及其对广泛的糖蛋白和糖缀合物的特异性，并与等效的植物来源的凝集素进行了比较。尽管 RPL 表现出更好或相似的特异性，但在所有情况下，它们的结合能力都明显更好。解释了结合机制，特别关注氢键在改变半乳糖特异性凝集素特异性方面的作用。此外，针对不同的糖蛋白靶标，还测试了不同的 RPL 及其植物等价物，以评估凝集素-糖蛋白相互作用的分析参数。除了一个之外，RPL 获得的 LoD 值都低于其植物对应物，对于针对岩藻糖、α-甘露糖、β-半乳糖和唾液酸的凝集素集合，RPL:PL LoD 比值分别为 0.8、2.5、14.2 和 380。RPL 分析参数的这种增强表明它们在蛋白质纯化以及作为聚糖分析和生物传感器开发的生物分析工具方面的适用性。
