Comparative analysis of fusion tags used to functionalize recombinant antibodies

Publication date: Available online 26 September 2019Source: Protein Expression and PurificationAuthor(s): Gianluca Veggiani, Barbara Giabbai, Marta S. Semrau, Barbara Medagli, Vincenzo Riccio, Gregor Bajc, Paola Storici, Ario de MarcoAbstractRecombinant 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.
Source: Protein Expression and Purification - Category: Biochemistry Source Type: research