(228ds) Silkworm Expression System Suitable for High-Throughput Screening of Antibody Fragment

Phage display method is the technique to select the antibodies with affinity for a target molecule such as membrane protein from the library of the phages displaying a large variety of antibody fragments on the surfaces. However, to identify the antibody with high binding affinity and structural stability, many antibody fragments as possible as we can select from the library should be prepared as recombinant protein to analyze the affinity and stability without phage, and the rapid protein preparation method for achieving the analysis should be prepared. Here, we focused on the protein production by means of silkworm. Because this protein production system can produce relatively large amount of proteins in small scale with high cellular density, it may prepare a variety of antibody fragments at a time for correct analysis. We develop process for preparing a variety of antibody fragments to obtain the antibody with affinity and stability.

We attempted to produce single chain fragments of variable region (scFv), where heavy chain of variable region (VH) and light chain of variable region (VL) are linked via a peptide linker. First, we inserted the scFv gene from the antibody with affinity for CD3 on lymphocyte cell and with affinity for epidermal growth factor receptor (EGFR) on cancer cells into baculovirus vectors and we transduced silkworm pupae with the vectors. The pupas were smashed and then ultracentrifuged. From the supernatant after ultracentrifugation, the scFv were purified by means of immobilized metal affinity chromatography and size exclusion chromatography. Finally, we measured the binding affinity of scFvs for each target molecule by means of flow cytometry and surface plasmon resonance.

In this study, 3 and 8 kinds of scFvs were prepared from anti-CD3 and anti-EGFR antibody, respectively. The scFvs were purified from the supernatant after ultracentrifugation, although the expressed amounts were varied. We confirmed that 8 out of 11 scFvs bound to each target molecule. From these results, we concluded that scFvs can be prepared from silkworms.