Production of scFv-conjugated affinity silk film and its application to a novel enzyme-linked immunosorbent assay

Abstract

Bombyx mori (silkworm) silk proteins have been utilized as unique biomaterials for various medical applications. To develop a novel affinity silk material, we generated a transgenic silkworm that spins silk protein containing the fibroin L-chain linked with the single-chain variable fragment (scFv) as a fusion protein. Previously, the scFv-conjugated “affinity” silk powder specifically immunoprecipitated its target protein, Wiskott-Aldrich syndrome protein. To expand the applicability of affinity silk materials, we processed the scFv-conjugated silk protein into a thin film by dissolving it in lithium bromide, then drying it in the wells of 96-well plates. Enzyme-linked immunosorbent assay demonstrated specific detection of Wiskott-Aldrich syndrome protein, both as a recombinant protein and in its native form extracted from mouse macrophages. These findings suggest that this scFv-conjugated silk film serves as the basis for an alternative immunodetection system.

Bioengineered silkworms with butterfly cytotoxin-modified silk glands produce sericin cocoons with a utility for a new biomaterial

“…The protein concentration in each sample was then measured using a Protein Quantification Assay Kit (Takara Bio). Protein samples (4 μg) were electrophoresed on an e-PAGEL 5-20% gradient gel (ATTO) and detected by Coomassie Brilliant Blue staining and by immunoblotting using an HRP-conjugated FibL-specific antibody (13).…”

“…Hence, the reduced weight of the cocoon shells made by w1-pnd P1A269/P1A269 larvae was considered most likely due to reduced fibroin content. SDS/PAGE in a gradient gel (5-20%) followed by immunoblotting with a FibL-specific antibody (13) showed that FibL and FibH proteins were present in the cocoon shells of w1-pnd +/+ larvae but not in the cocoons shells of w1-pnd P1A269/P1A269 larvae, in which only nonfibroin proteins, mainly sericins (15,(19)(20)(21)(22), were detectable (sericin 1-4; Fig. 2C).…”

“…Accordingly, transgenic silkworms have been studied as tools for large-scale production of foreign recombinant proteins because silkworm silk glands have a high capacity for protein biosynthesis (12)(13)(14). Silk proteins from silkworm cocoons, which are mainly composed of fibroin and sericin, are candidate biomaterials with numerous biomedical applications (15,16).…”

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

“…The scFvconjugated silk fibroin showed antigen binding capacity when used as a capture probe in enzyme-linked immuno sorbent assay (ELISA). However, production using the transgenic silkworm technology generally yields low levels of target protein with a majority of endogenous silk worm fibroin [21], and tedious post processing is needed in case other formats than silk cocoons are wanted [20,21].…”

Incorporation of Methionine Analogues Into Bombyx mori Silk Fibroin for Click Modifications

“…To date, genetic engineering and chemical modification have been employed to alter the characteristics of silk‐based materials. In particular, genetically engineered silk has been emerging as a resource for the development of functionally and mechanically high‐performance biomaterials.…”