Production of scFv-Conjugated Affinity Silk Powder by Transgenic Silkworm Technology

Sato, Mitsuru; Kojima, Katsura; Sakuma, Chisato; Murakami, Maria; Aratani, Eriko; Takenouchi, Takato; Tamada, Yasushi; Kitani, Hiroshi

doi:10.1371/journal.pone.0034632

Cited by 23 publications

(42 citation statements)

References 28 publications

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“…2a) that were produced and affinity-purified from Escherichia coli cells23. GST-WASP15 is the specific antigen against which the parental anti-WASP mAb1922 was raised.…”

Section: Resultsmentioning

confidence: 99%

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

Sato

Kojima

Sakuma

et al. 2014

Sci Rep

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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.

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“…2a) that were produced and affinity-purified from Escherichia coli cells23. GST-WASP15 is the specific antigen against which the parental anti-WASP mAb1922 was raised.…”

Section: Resultsmentioning

confidence: 99%

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

Sato

Kojima

Sakuma

et al. 2014

Sci Rep

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“…it was fabricated as powder form and its specific antigen-binding capability was confirmed [29]. The "affinity silk" was defined as a silk which has a modified FibL protein fused with a single-chain fragment variable (scFv), instead of the whole IgG protein, at the C-terminus and is expressed as a part of silk fibroin (Fig.…”

Section: Affinity Silkmentioning

confidence: 99%

Silk Fibers as Smart Materials Toward Medical Textiles

Tamada¹,

Kojima²

2014

Handbook of Smart Textiles

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Silk has been used as a textile fiber for more than 6,000 years. Recently, many applications of silk have been studied for use beyond textiles, especially in medical areas. Silk is reported as a safe material with excellent mechanical properties and biocompatibility. In 2000, a transgenic silkworm technology developed based on the transposon piggyBac and microinjection technique, producing transgenic silk fibers having additional new functions to silk, became available. It was expected to expand the application area of silk into medical and other fields. Herein, silk fiber and studies of silk use in medical applications are described, along with an introduction of transgenic silkworm technologies used to produce a smart material based on silk. Cell-adhesive silk fiber with celladhesive peptide "RGDS" will be used for cell scaffolds in tissue engineering. "Affinity silk," with a fused single-chain fragment variable (scFv) of IgG protein to silk fibroin protein, will be valuable for affinity purification systems, diagnosis of diseases, detection of pathogenic microorganisms, and development of therapeutic strategies. Spider silk will become an engineering fiber with excellent mechanical strength from a natural origin. Silk fibers are anticipated for use as a smart material in advanced textiles intended for use in medical and various other fields.

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“…However, practice of this means of immobilization is limited to the non-covalent type of interaction between histidine tagged scFv and solid support coated with divalent metal ions, which might give lower performance duration compared to the covalent way of immobilization [6,19]. In a recent study, scFv directed against Wiskott-Aldrich syndrome protein (WASP) was conjugated to silk worm fibroin and produced as a fusion protein in transgenic silkworm [20]. The scFvconjugated silk fibroin showed antigen binding capacity when used as a capture probe in enzyme-linked immuno sorbent assay (ELISA).…”

Section: Introductionmentioning

confidence: 99%

“…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].…”

Section: Introductionmentioning

confidence: 99%

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

Thatikonda

Delfani

Jansson

et al. 2015

Biotechnology Journal

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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.

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Production of scFv-Conjugated Affinity Silk Powder by Transgenic Silkworm Technology

Cited by 23 publications

References 28 publications

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

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

Silk Fibers as Smart Materials Toward Medical Textiles

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

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