A 33-residue peptide tag increases solubility and stability of Escherichia coli produced single-chain antibody fragments

Abstract

Single-chain variable fragments (scFvs), composed of variable domains of heavy and light chains of an antibody joined by a linker, share antigen binding capacity with their parental antibody. Due to intrinsically low solubility and stability, only two Escherichia coli-produced scFvs have been approved for therapy. Here we report that a 33-residue peptide, termed P17 tag, increases the solubility of multiple scFvs produced in Escherichia coli SHuffle strain by up to 11.6 fold. Hydrophilic sequence, especially charged residues, but not the predicted α-helical secondary structure of P17 tag, contribute to the solubility enhancement. Notably, the P17 tag elevates the thermostability of scFv as efficiently as intra-domain disulfide bonds. Moreover, a P17-tagged scFv targeting hepatitis B virus surface proteins shows over two-fold higher antigen-binding affinity and virus-neutralizing activity than the untagged version. These data strongly suggest a type I intramolecular chaperone-like activity of the P17 tag. Hence, the P17 tag could benefit the research, production, and application of scFv.

Food-Borne Biotoxin Neutralization in Vivo by Nanobodies: Current Status and Prospects

“…Genetic modifications and optimized cultivation conditions can still mitigate these effects . Currently, these effective modification methods are being explored, including optimizing linkers, ferritin fusion, and the use of soluble tags . Overall, with the continuous progress and optimization of production techniques, the efficiency and quality of Nbs and multivalent Nbs production will further increase, providing strong support for their commercial applications.…”

“…Striking this balance holds the key to fully harnessing the advantages inherent in multivalent Nbs for practical applications. Research has found that the fusion of ferritin skeletons, the use of soluble tags, and the optimization of elements such as promoters and signal peptides can effectively enhance the overall expression yield of Nbs. , These studies will provide strong support for the efficient production and commercial application of Nbs and multivalent Nbs.…”

Improvement of the sensitivity of lateral flow systems for detecting mycotoxins: Up‐to‐date strategies and future perspectives

“…Wang, Yuan, et al. (2022) reported that a P17 peptide tag improved the solubility, stability, and affinity of single‐chain antibodies.…”

“…In a recent study, the introduction of peptide tags into single-chain antibodies has provided another strategy to improve sensitivity. Wang, Yuan, et al (2022) reported that a P17 peptide tag improved the solubility, stability, and affinity of single-chain antibodies.…”

Identification of an N-terminal tag (580N) that improves the biosynthesis of fluorescent proteins in Francisella tularensis and other Gram-negative bacteria

“…Even with the development of molecular tools and controllable promotor systems, the expression of recombinant proteins by bacteria can still be sub-optimal. One strategy to overcome meager recombinant protein expression is to utilize a peptide tag fusion [ 19 – 21 ]. For instance, the linkage of the secretory signal peptide from a Bacillus thuringiensis insecticidal protein (Cry1Ia; Iasp) to eGFP (enhanced GFP) led to increased expression of this fluorescent protein [ 22 ].…”