Abstract
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.
Highlights
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)
The amino acid sequence RGDSPASSK of the FG loop region of FN3 was replaced by epitope 12–21 of NT-proBNP
The epitope 62–73 of NT-proBNP was recombined with the C-terminus of FN3, and the linker KKGKGKKGK was added to facilitate coupling with other molecules, such as Biotin and peroxidase
Summary
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). The structure of the three flexible surface-exposed loops (BC, DE, and FG) of FN3 are similar to that of antibody variable districts, and the change in amino acid sequences in the BC and FG loops did not disturb the stability of macromolecular derivatives of FN3 (Koide et al, 1998; Liu et al, 2019). We efficiently expressed monobodies and receptor proteins based on FN3 as a protein scaffold in E. coli (Ding et al, 2019; Zhu et al, 2020)
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
