Comparative analysis of fusion tags used to functionalize recombinant antibodies

Veggiani, Gianluca; Giabbai, Barbara; Semrau, Marta Stefania; Medagli, Barbara; Riccio, Vincenzo; Bajc, Gregor; Storici, Paola; Marco, Ario de

doi:10.1016/j.pep.2019.105505

Cited by 12 publications

(7 citation statements)

References 37 publications

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“…However, a large number of nanobodies are currently mainly produced in E. coli cells with a low yield. Although several fusion tags have been used to functionalize nanobodies (Gotzke et al 2019;Veggiani et al 2020), few of them demonstrated an expressing enhancement effect on nanobodies. Previously, we found an interesting result that the expression of nanobodies was enhanced by the fusion with an SFP hexapeptide (GAGAGS).…”

Section: Discussionmentioning

confidence: 99%

A novel silk fibroin protein–based fusion system for enhancing the expression of nanobodies in Escherichia coli

Yang

et al. 2022

Appl Microbiol Biotechnol

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Nanobodies show a great potential in biomedical and biotechnology applications. Bacterial expression is the most widely used expression system for nanobody production. However, the yield of nanobodies is relatively low compared to that of eukaryotic systems. In this study, the repetitive amino acid sequence motifs (GAGAGS) found in silk fibroin protein (SFP) were developed as a novel fusion tag (SF-tag) to enhance the expression of nanobodies in Escherichia coli. SF-tags of 1 to 5 hexapeptide units were fused to the C-terminus of 4G8, a nanobody against human epididymis protein 4 (HE4). The protein yield of 4G8 variants was increased by the extension of hexapeptide units and achieved a 2.5 ~ 7.1-fold increase compared with that of untagged 4G8 (protein yield of 4G8-5C = 0.307 mg/g vs that of untagged 4G8 = 0.043 mg/g). Moreover, the fusion of SF-tags not only had no significant effect on the affinity of 4G8, but also showed a slight increase in the thermal stability of SF-tag-fused 4G8 variants. The fusion of SF-tags increased the transcription of 4G8 by 2.3 ~ 7.0-fold, indicating SF-tags enhanced the protein expression at the transcriptional level. To verify the applicability of the SF-tags for other nanobody expression, we further investigated the protein expression of two other anti-HE4 nanobodies 1G8 and 3A3 upon fusion with the SF-tags. Results indicated that the SF-tags enhanced the protein expression up to 5.2-fold and 5.7-fold for 1G8 and 3A3, respectively. For the first time, this study reported a novel and versatile fusion tag system based on the SFP for improving nanobody expression in Escherichia coli, which may enhance its potential for wider applications. Key points • A silk fibroin protein-based fusion tag (SF-tag) was developed to enhance the expression of nanobodies in Escherichia coli.• The SF-tag enhanced the nanobody expression at the transcriptional level.• The fusion of SF-tag had no significant effect on the affinity of nanobodies and could slightly increase the thermal stability of nanobodies.

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Section: Discussionmentioning

confidence: 99%

A novel silk fibroin protein–based fusion system for enhancing the expression of nanobodies in Escherichia coli

Yang

et al. 2022

Appl Microbiol Biotechnol

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“…The expression of nanobodies and, more and more frequently, nanobodies fused to tags offering orthogonal functions, becomes constantly more diversified [32,130]. This is necessary to obtain reagents that possess the highly differentiated features necessary for fulfilling the always new final application requirements and consequently their production relies on alternative expression conditions [47].…”

Section: Discussionmentioning

confidence: 99%

“…When the sequence of a nanobody is available after panning, its expression should be planned considering the final application of the resulting construct. We developed and validated a large array of modular vectors based on pET vector scaffolds that share: i) a conserved cloning site for the nanobody sequence (NcoI/NotI); ii) a further cloning cassette for inserting a "functional tag" such as fluorescent proteins, Avitag, SNAP, free cysteine, SpyTag …; iii) a poly-His tag for affinity purification [13,44,47,62,[126][127][128][129][130][131]. This concept enables to exchange the modules and adapt existing constructs to design new vector versions.…”

Section: Practical Advice For Nanobody Expression and Purificationmentioning

confidence: 99%

Recombinant expression of nanobodies and nanobody-derived immunoreagents

Marco

2020

Protein Expression and Purification

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A B S T R A C TAntibody fragments for which the sequence is available are suitable for straightforward engineering and expression in both eukaryotic and prokaryotic systems. When produced as fusions with convenient tags, they become reagents which pair their selective binding capacity to an orthogonal function. Several kinds of immunoreagents composed by nanobodies and either large proteins or short sequences have been designed for providing inexpensive ready-to-use biological tools. The possibility to choose among alternative expression strategies is critical because the fusion moieties might require specific conditions for correct folding or posttranslational modifications. In the case of nanobody production, the trend is towards simpler but reliable (bacterial) methods that can substitute for more cumbersome processes requiring the use of eukaryotic systems. The use of these will not disappear, but will be restricted to those cases in which the final immunoconstructs must have features that cannot be obtained in prokaryotic cells. At the same time, bacterial expression has evolved from the conventional procedure which considered exclusively the nanobody and nanobody-fusion accumulation in the periplasm. Several reports show the advantage of cytoplasmic expression, surface-display and secretion for at least some applications. Finally, there is an increasing interest to use as a model the short nanobody sequence for the development of in silico methodologies aimed at optimizing the yields, stability and affinity of recombinant antibodies.

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“…Sortase-mediated ligation (SML) is a powerful biomolecular tool for the post-translational modification of a target protein. Sortase A is a transpeptidase that is natively found in Gram-positive bacteria, where it is responsible for anchoring proteins onto the bacterial membrane with small peptide tags. − The most commonly used sortase enzyme found in Staphylococcus aureus catalyzes a peptide bond between an LPXTG motif (where X is any amino acid), commonly placed near the C-terminus of a protein substrate, and an N-terminal G n motif-containing partner (Figure A). ,, Because sortase processing requires only small peptide tags, SML in proteins is useful for incorporating a broad range of moieties ranging from synthetic small molecules − to biopolymers − to conventional peptides ,, and proteins , (Figure B). Furthermore, adding separate recognition motifs at the N- and C-terminus of the target protein can facilitate multiple modifications , by using orthogonal sortases, such as those found in Streptococcus pyogenes , ,,, Lactobacillus plantarum , or derivative mutants from S. aureus.…”

Section: Sortase-mediated Ligationmentioning

confidence: 99%

Site-Specific Bioconjugation Approaches for Enhanced Delivery of Protein Therapeutics and Protein Drug Carriers

et al. 2020

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Proteins have the capacity to treat a multitude of diseases both as therapeutics and as drug carriers due to their complex functional properties, specificity toward binding partners, biocompatibility, and programmability. Despite this, native proteins often require assistance to target diseased tissue due to poor pharmacokinetic properties and membrane impermeability. Functionalizing therapeutic proteins and drug carriers through direct conjugation of delivery moieties can enhance delivery capabilities. Traditionally, this has been accomplished through bioconjugation methods that have little control over the location or orientation of the modification, leading to highly heterogeneous products with varying activity. A multitude of promising site-specific protein conjugation methods have been developed to allow more tailorable display of delivery moieties and thereby enhance protein activity, circulation properties, and targeting specificity. Here, we focus on three particularly promising site-specific bioconjugation techniques for protein delivery: unnatural amino acid incorporation, Sortase-mediated ligation, and SpyCatcher/SpyTag chemistry. In this review, we highlight the promise of site-specific bioconjugation for targeted drug delivery by summarizing impactful examples in literature, considering important design principles when constructing bioconjugates, and discussing our perspectives on future directions.

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Comparative analysis of fusion tags used to functionalize recombinant antibodies

Cited by 12 publications

References 37 publications

A novel silk fibroin protein–based fusion system for enhancing the expression of nanobodies in Escherichia coli

A novel silk fibroin protein–based fusion system for enhancing the expression of nanobodies in Escherichia coli

Recombinant expression of nanobodies and nanobody-derived immunoreagents

Site-Specific Bioconjugation Approaches for Enhanced Delivery of Protein Therapeutics and Protein Drug Carriers

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