Construction of Artificial TNF-Binding Proteins Based on the 10th Human Fibronectin Type III Domain Using Bacterial Display

Shingarova, L. N.; Petrovskaya, L. E.; Zlobinov, Alexander V.; Gapizov, Sultan Sh.; Kryukova, E. A.; Birikh, Klara R.; Boldyreva, E. F.; Yakimov, S. A.; Долгих, Д. А.; Kirpichnikov, Mikhaǐl P.

doi:10.1134/s0006297918060081

Cited by 6 publications

(2 citation statements)

References 37 publications

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“…These proteins are often used for engineering synthetic binding proteins due to their high stability, low molecular weight and are easy to produce using bacterial expression systems. Some view this approach as a replacement for antibody therapy [76,77]. For example, ACPs have been developed based on the 10th FN3 domain of human fibronectin for binding the cancer biomarker mesothelin [78].…”

Section: Fn3: Species Specificity Divergence Structure Functionmentioning

confidence: 99%

The Role of the PFNA Operon of Bifidobacteria in the Recognition of Host’s Immune Signals: Prospects for the Use of the FN3 Protein in the Treatment of COVID-19

Nezametdinova

Yunes

Dukhinova

et al. 2021

IJMS

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Bifidobacteria are some of the major agents that shaped the immune system of many members of the animal kingdom during their evolution. Over recent years, the question of concrete mechanisms underlying the immunomodulatory properties of bifidobacteria has been addressed in both animal and human studies. A possible candidate for this role has been discovered recently. The PFNA cluster, consisting of five core genes, pkb2, fn3, aaa-atp, duf58, tgm, has been found in all gut-dwelling autochthonous bifidobacterial species of humans. The sensory region of the species-specific serine-threonine protein kinase (PKB2), the transmembrane region of the microbial transglutaminase (TGM), and the type-III fibronectin domain-containing protein (FN3) encoded by the I gene imply that the PFNA cluster might be implicated in the interaction between bacteria and the host immune system. Moreover, the FN3 protein encoded by one of the genes making up the PFNA cluster, contains domains and motifs of cytokine receptors capable of selectively binding TNF-α. The PFNA cluster could play an important role for sensing signals of the immune system. Among the practical implications of this finding is the creation of anti-inflammatory drugs aimed at alleviating cytokine storms, one of the dire consequences resulting from SARS-CoV-2 infection.

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Section: Fn3: Species Specificity Divergence Structure Functionmentioning

confidence: 99%

The Role of the PFNA Operon of Bifidobacteria in the Recognition of Host’s Immune Signals: Prospects for the Use of the FN3 Protein in the Treatment of COVID-19

Nezametdinova

Yunes

Dukhinova

et al. 2021

IJMS

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“…The 10th human fibronectin type III (FN3) domain has become one of the most widely used nonantibody scaffolds for engineering novel binding proteins (Koide et al, 1998;Gill and Damle, 2006;Chandler and Buckle, 2020). Several FN3-based biological molecules have been developed for therapeutic and diagnostic applications (Shingarova et al, 2018;Chandler and Buckle, 2020;Sirois et al, 2020). The structural simplicity and superior biophysical characteristics of FN3 offer advantages for the construction of stable and novel biomacromolecules.…”

Section: Introductionmentioning

confidence: 99%

FN3 Domain Displaying Double Epitopes: A Cost-Effective Strategy for Producing Substitute Antigens

Ruan

Chao

et al. 2021

Front. Mol. Biosci.

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Construction of substitute antigens based on alternative scaffold proteins is a promising strategy in bioassay technology. In this study, we proposed a strategy for constructing substitute antigens derived from 10th human fibronectin type III (FN3) using two peptide epitopes of terminal pro-brain natriuretic peptide (NT-proBNP) as an example. The base sequences encoding the two antigenic epitopes of NT-proBNP were recombined into the FG loop region and the C-terminus of FN3, fused by 4 GS or polyN linker. The fusion proteins (named FN3-epitopes-4GS and FN3-epitopes-polyN, respectively) were expressed and purified cost-effectively using an Escherichia coli expression system. The immunoreactivity of recombinant substitutes was preliminarily confirmed by western blot analysis using epitope-specific antibodies. The sandwich enzyme-linked immunosorbent assay demonstrated that either FN3-epitopes-polyN or FN3-epitopes-4GS was highly sensitive, and FN3-epitopes-polyN exhibited better kinetics to specific antibodies than FN3-epitopes-4GS, showing a linear dose-response relationship in the concentration range of 0.06–12.85 ng/ml, which suggest that the polyN linker was more suitable for constructing the FN3-based substitute antigens compared to the 4 GS linker. Furthermore, the serum stability test and differential scanning calorimetry analysis showed that the recombinant FN3-epitopes-polyN maintained the original stability of FN3. Therefore, it was confirmed that FN3 could be engineered to construct a stable biomacromolecular substitute for displaying double epitopes of antigen proteins, such as NT-proBNP. In summary, a cost-effective strategy to produce NT-proBNP substitute antigens with good immunoreactivity and physicochemical stability was established in this work, which may provide potential uses for the production of other substitute antigens in the future.

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Engineering of binding functions into proteins

Gebauer¹,

Skerra

2019

Current Opinion in Biotechnology

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Construction of Artificial TNF-Binding Proteins Based on the 10th Human Fibronectin Type III Domain Using Bacterial Display

Cited by 6 publications

References 37 publications

The Role of the PFNA Operon of Bifidobacteria in the Recognition of Host’s Immune Signals: Prospects for the Use of the FN3 Protein in the Treatment of COVID-19

The Role of the PFNA Operon of Bifidobacteria in the Recognition of Host’s Immune Signals: Prospects for the Use of the FN3 Protein in the Treatment of COVID-19

FN3 Domain Displaying Double Epitopes: A Cost-Effective Strategy for Producing Substitute Antigens

Engineering of binding functions into proteins

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