Mitsuru Sato¹, Katsura Kojima², Yasushi Tamada², Hiroshi Kitani¹

¹Animal Immune and Cell Biology Research Unit, ²Silk Materials Research Unit

［Abstract］

Bombyx mori (silkworm) silk proteins are being utilized as unique biomaterials for medical applications. Chemical modification or post-conjugation of bioactive ligands expands the applicability of silk proteins; however, the processes are elaborate and costly. In this study, we used transgenic silkworm technology to develop single-chain variable fragment (scFv)-conjugated silk fibroin as a novel affinity material.

［Keywords］

transgenic silkworm, single-chain variable fragment, affinity silk powder

［Background］

Bombyx mori (silkworm) silk has been recognized as a unique natural biopolymer for various biomedical applications. After silk fibers are dissolved in aqueous solution, this protein can be fabricated into various material formats such as powder, fiber, gel, sponge, or thin film. Recent advances in transgenic silkworm technology have demonstrated that recombinant proteins can be produced in the silk glands, either independently from the silk proteins, or fused with fibroin proteins, which retain their original structure and function. To expand the applicability of transgenic silk fibroins as a novel affinity reagent, we generated a transgenic silkworm strain that produces silk fibroin protein fused to singlechain variable fragment (scFv), which is composed of V_H and V_L domains from the original antibody. The present work demonstrates the promising possibility of scFv-conjugated silk fibroin proteins as a unique alternative to conventional affinity reagents.

［Results and Discussion］

1. We used transgenic silkworm technology to develop scFv-conjugated silk fibroin. The cocoons of the transgenic silkworm contain fibroin L-chain linked with scFv as a fusion protein (Fig.1).
2. The scFv construct was derived from a monoclonal antibody (mAb) against Wiskott-Aldrich syndrome protein (WASP), which is an important immune adaptor molecule in mammals.
3. After dissolving the cocoons in 9M LiBr, the silk solution was dialyzed, concentrated, freeze-dried, and crushed into powder.
4. Anti-WASP-scFv-conjugated silk powder has equivalent immunoprecipitation potency compared with its parental mAb-coupled protein G-sepharose (Fig. 2).
5. Given these observations, silk powder made from cocoons expressing scFv fused to fibroin protein may be potentially useful for developing an alternative reagent that retains affinity to target proteins comparable to that of conventional immunoprecipitation reagents.

1. Affinity silk powders are ready-made reagents produced by transgenic silkworm technology, and require only a few purification steps. Therefore, affinity silk powders can be manufactured at a lower cost than those associated with traditional affinity carriers.
2. Further improvement of affinity silk technologies would provide novel materials that can be applied to the development of an affinity purification system, the diagnosis of diseases, and the detection of pathogenic microorganisms.

Fig.1. Transgenic silkworms produce genetically engineered fibroin protein in the silk fibers.

Fig.2. Affinity purification of the target molecule using affinity silk powder. A mouse macrophage RAW 264.7 cell line was lysed and incubated separately with silk powder particle from wild-type or transgenic strains, or with protein G-sepharose coupled with anti-WASP mAb or control mouse IgG. Immunocomplexes were analyzed by Western blotting with anti-WASP pAb.

 

[Reference]

1. Sato M, Kojima K, Sakuma C, Murakami M, Aratani E, Takenouchi T, Tamada Y, Kitani H (2012) Production of scFv-conjugated affinity silk powder by transgenic silkworm technology PLoS ONE 7(4):e34632.
2. Murakami M, Tamada Y, Kojima K (2012) Dissolving whole cocoon silk proteins by using a pHadjusted buffered LiBr solution Nippon Silk Gakkaishi 20:89-94. (In Japanese)
3. Patent application# JP-2012-239436 (Japan)