Production of a recombinant mouse monoclonal antibody in transgenic silkworm cocoons.
In the present study, we describe the production of transgenic silkworms expressing a recombinant mouse mAb in their cocoons. Two transgenic lines, L- and H-, were generated that carried cDNAs encoding the L- and H-chains of a mouse IgG mAb, respectively, under the control of the enhancer-linked sericin-1 promoter. Cocoon protein analysis indicated that the IgG L- or H-chain was secreted into the cocoons of each line. We also produced a transgenic line designated L/H, which carried both cDNAs, by crossing the L- and H-lines. This line efficiently produced the recombinant mAb as a fully assembled H(2)L(2) tetramer in its cocoons, with negligible L- or H-chain monomer and H-chain dimer production. Thus, the H(2)L(2) tetramer was synthesized in, and secreted from, the middle silk gland cells. Crossing of the L/H-line with a transgenic line expressing a baculovirus-derived trans-activator produced a 2.4-fold increase in mAb expression. The recombinant mAb was extracted from the cocoons with a buffer containing 3 m urea and purified by protein G affinity column chromatography. The antigen-binding affinity of the purified recombinant mAb was identical to that of the native mAb produced by a hybridoma. Analysis of the structure of the N-glycans attached to the recombinant mAb revealed that the mAb contained high mannose-, hybrid- and complex-type N-glycans. By contrast, insect-specific paucimannose-type glycans were not detected. Fucose residues alpha-1,3- and alpha-1,6-linked to the core N-acetylglucosamine residue, both of which are found in insect N-glycans, were not observed in the N-glycans of the mAb.
Iizuka
Masashi
M
Neosilk Co., Ltd, Higashihiroshima, Hiroshima, Japan.
Ogawa
Shingo
S
Takeuchi
Atsushi
A
Nakakita
Shinichi
S
Kubo
Yuhki
Y
Miyawaki
Yoshitaka
Y
Hirabayashi
Jun
J
Tomita
Masahiro
M
D000818:Animals; D030801:Animals, Genetically Modified / Q000235:genetics / Q000378:metabolism; D000888:Antibodies, Anti-Idiotypic / Q000096:biosynthesis* / Q000235:genetics; D000911:Antibodies, Monoclonal / Q000096:biosynthesis* / Q000235:genetics; D012831:Bombyx / Q000235:genetics / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D051379:Mice; D011401:Promoter Regions, Genetic / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics; D047030:Sericins / Q000235:genetics; D047011:Silk / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D000888:Antibodies, Anti-Idiotypic; D000911:Antibodies, Monoclonal; D011993:Recombinant Fusion Proteins; D047030:Sericins; D047011:Silk; C048844:anti-IgG