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Biotechnology and Applied Biochemistry (1999) 30, (59–64) (Printed in Great Britain)

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Expression and purification of a secreted functionalmouse/human chimaeric antibody against bacterial endotoxin inbaculovirus-infected insect cells

Weiqi Tan*¹ and Peter H. Y. Lam†

*National Laboratory of Biomembrane and MembraneBiotechnology, Institute of Zoology, Academia Sinica, Beijing100080, People's Republic of China, and †Department ofBiochemistry, Hong Kong University of Science and Technology,Hong Kong, People's Republic of China

Abbreviations used: Fab, antigen-binding fragment; Fc, crystallizing fragment; Fd, heavy-chain fragment V_HC_H1; HAMA, human anti-mouse antibody; HC, heavy-chain gene; LC, light-chain gene; mAb, monoclonal antibody; PNGase F, peptide N-glycosidase F.

¹ To whom correspondence should be addressed.

We have created a mouse/human chimaeric antibody by taking antigen-binding fragment (Fab) genes of a mouse antibody-producing hybridoma with specificity for bacterial endotoxin and joining them to human Ig crystallizing-fragment (Fc) genes using recombinant DNA techniques. This chimaeric antibody has been expressed in Sf21 and High Five™ (BTI-TN-5B1-4) insect cells using the baculovirus expression system, which may allow the mass production of secretory recombinant antibodies. This was achieved by using infection with a double-recombinant virus containing cDNAs of both the Ig heavy-chain (HC) and light-chain (LC) genes. Prior to recombination, each gene was cloned into the dual-expression baculovirus transfer vector pPLSP2, which permitted the insertion of the LC gene in-frame with the signal peptide of honey bee melittin downstream of the polyhedrin promoter, and of the HC gene in-frame with the signal peptide of Bombyx mori larval serum protein downstream of the p10 promoter. Our results showed that the polypeptide chains were secreted by insect cells and correctly assembled into H₂L₂ heterodimers containing N-linked carbohydrate at the heavy chain. Furthermore, the recombinant chimaeric antibody exhibited a similar antigen specificity to that of the monoclonal antibody. More importantly, it provides a generic method for the high-level expression of antibodies.

Received 10 March 1999/16 April 1999; accepted 4 May 1999

Portland Press Ltd © 1999