Recombinant antibodies can be expressed as fusion constructs in combination with tags which simplify their engineering into reliable and homogeneous immunoreagents by allowing site-specific, 1:1 functionalization. Several tags and corresponding reagents for recombinant protein derivatization have been proposed but benchmarking surveys for the evaluation of their effect on the characteristics of recombinant antibodies have not been reported. In this work we evaluated the impact on expression yields, shelf-stability, thermostability and binding affinity of a set of C-terminal tags fused to the same anti-Her2 nanobody. Furthermore, we assessed the efficiency of the derivatization process. The constructs always bore a 6xHis tag plus either the controls (EGFP and C-tag) or CLIP, HALO, AviTag, the LEPTG sequence recognized by Sortase A (Sortase tag), or a free cysteine. The advantages and drawbacks of the different systems were analyzed and discussed.

中文翻译：

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用于功能化重组抗体的融合标签的比较分析。

重组抗体可与标签一起表达为融合构建体，通过允许位点特异性，1：1功能化，简化了工程化为可靠且均质的免疫试剂的过程。已经提出了几种用于重组蛋白衍生化的标签和相应试剂，但是尚未报道用于评估其对重组抗体特性影响的基准研究。在这项工作中，我们评估了融合到相同抗Her2纳米抗体上的一组C末端标签对表达产量，货架稳定性，热稳定性和结合亲和力的影响。此外，我们评估了衍生化过程的效率。这些构建体始终带有6xHis标签以及对照（EGFP和C标签）或CLIP，HALO，AviTag，Sortase A识别的LEPTG序列（Sortase标签），或游离的半胱氨酸。分析并讨论了不同系统的优缺点。