Triton X-114 和基于胺的洗涤策略将脂多糖减少到 FDA 限制并获得更纯净、更有效的重组免疫毒素,Bioconjugate Chemistry

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Triton X-114 和基于胺的洗涤策略将脂多糖减少到 FDA 限制并获得更纯净、更有效的重组免疫毒素
Bioconjugate Chemistry ( IF 4.0 ) Pub Date : 2021-04-01 , DOI: 10.1021/acs.bioconjchem.1c00013
Rebekka George _{1,

2} , Franziska Gsottberger ₁ , Anna Ammon ₁ , Kerstin Wendland ₁ , Lisa Mellenthin ₁ , Andreas Mackensen ₁ , Fabian Müller ₁

Affiliation

许多蛋白质仍然在大肠杆菌中常规原核表达，其中一些是因为它们对真核生物有毒。免疫毒素是靶向部分和截短假单胞菌外毒素 A 的融合蛋白，通过阻止蛋白质合成杀死靶细胞。因此，免疫毒素必须在大肠杆菌中表达。在大肠杆菌中表达的蛋白质被内毒素（也称为脂多糖 (LPS)）污染。LPS 与 toll 样受体结合，在哺乳动物中诱发危及生命的全身炎症。因此，对于治疗剂以及动物免疫学研究中使用的物质，公认的 LPS 限值非常低。在这里，我们报告了使用 Triton X-114 和基于多胺的洗涤策略，只有结合使用才能实现远低于 FDA 限制的 LPS 污染。由此产生的 LPS 减少的免疫毒素更纯，体外活性高达 2.4 倍。增加的活性与细胞表面表达的靶抗原的亲和力增加 2.4 倍有关。该组合方法保持了酶促功能、蛋白质稳定性和体内功效，并且对 Fab 和 dsFv 格式有效。通过一些修改，这种新型组合的原理可以应用于任何基于色谱的纯化过程。

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Triton X-114 and Amine-Based Wash Strategy Reduces Lipopolysaccharides to FDA Limit and Achieves Purer, More Potent Recombinant Immunotoxin

Many proteins are still routinely expressed prokaryotically in Escherichia coli, some because they are toxic to eukaryotes. Immunotoxins, which are fusion proteins of a targeting moiety and a truncated Pseudomonas exotoxin A, kill target cells by arresting protein synthesis. Thus, immunotoxins must be expressed in E. coli. Proteins expressed in E. coli are contaminated by endotoxin (also called lipopolysaccharides (LPS)). LPS binds to toll-like receptors, inducing up to life-threatening systemic inflammation in mammals. Therefore, accepted LPS limits for therapeutics as well as for substances used in immunological studies in animals are very low. Here, we report the use of Triton X-114 and polyamine-based wash strategies, which only in combination achieved LPS-contamination well below FDA limits. Resulting LPS-reduced immunotoxins were purer and up to 2.4-fold more active in vitro. Increased activity was associated with a 2.4-fold increase in affinity on cell surface expressed target antigen. The combination method maintained enzymatic function, protein stability, and in vivo efficacy and was effective for Fab as well as dsFv formats. With some modifications, the principle of this novel combination may be applied to any chromatography-based purification process.

更新日期：2021-04-21

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