Genetic fusion of single‐chain variable fragments to partial spider silk improves target detection in micro‐ and nanoarrays

Abstract

Immobilizing biomolecules with retained functionality and stability on solid supports is crucial for generation of sensitive immunoassays. However, upon use of conventional immobilization strategies, a major portion of the biomolecules (e.g. antibodies) frequently tends to lose their bioactivity. In this study, we describe a procedure to immobilize human single-chain variable fragment (scFv) via genetic fusion to partial spider silk, which have a high tendency to adhere to solid supports. Two scFvs, directed towards serum proteins, were genetically fused to partial spider silk proteins and expressed as silk fusion proteins in E. coli. Antigen binding ability of scFvs attached to a partial silk protein denoted RC was investigated using microarray analysis, whereas scFvs fused to the NC silk variant were examined using nanoarrays. Results from micro- and nanoarrays confirmed the functionality of scFvs attached to both RC and NC silk, and also for binding of targets in crude serum. Furthermore, the same amount of added scFv gives higher signal intensity when immobilized via partial spider silk compared to when immobilized alone. Together, the results suggest that usage of scFv-silk fusion proteins in immunoassays could improve target detection, in the long run enabling novel biomarkers to be detected in crude serum proteomes.

Bioactivation of Spider Silk with Basic Fibroblast Growth Factor for in Vitro Cell Culture: A Step toward Creation of Artificial ECM

“…affinity domains , and enzyme were successfully incorporated to WT-silk using recombinant DNA technology. These incorporated protein domains retained their biological activity and did not interfere with the silk-assembly properties of WT-silk. ,, However, in all earlier studies, the protein domains were conjugated to the N-terminus of WT-silk. ,, In this study, genetic fusion of bFGF to the N-terminus of WT-silk resulted in a low protein yield (data not shown), and for this reason bFGF fused to C-terminus of WT-silk was used instead. A glycine-serine rich linker was incorporated between WT-silk and bFGF with the aim of improving availability of bFGF after its conjugation to silk (Figure a).…”

“…However, the possibility to control orientation and distribution of such entrapped growth factors is limited . Dense and oriented immobilization of affinity domains has been shown to be possible by covalent attachment to recombinant spider silk, 4RepCT at the gene level, , and a similar strategy would thus likely be suitable also for GFs. Another common strategy for covalent immobilization of GFs is based on carbodiimide-mediated conjugation, which requires the presence of available primary amines or carboxylate functional groups on the GFs .…”

Site-selective orientated immobilization of antibodies and conjugates for immunodiagnostics development

“…[181] For example, a polystyrene-binding peptide (PS-tag) fused V H H was constructed and used for ELISA,[42] where PS-Tag facilitated not only adsorption but also orientated adsorption on PS support via hydrophobic forces. [42,182–184] Besides PS-tag, PMMA- [185] and spider silk protein [186] tags have been reported as fusion proteins to recombinant antibodies for orientated immobilization on corresponding solid supports.…”

Silk Materials Functionalized via Genetic Engineering for Biomedical Applications

“…Thatikonnda et al proposed a silk-based material composed of silk fused with a single-chain variable fragment (scFv) derived from a human antibody [ 111 ]. The scFv combines variable fragments of light and heavy antibody chains—the parts responsible for antigen recognition.…”

“…Interestingly, the fusion of scFv domains did not impair silks processing into fibers, especially taking into consideration the similar size of the fused components. Moreover, the scFv/NC variants indicated better solubility properties that allowed nanodispensing of these silks into nanoarrays [ 111 ]. This kind of material appears to be a promising candidate for next-generation immunoassays and biosensors.…”