Expression and secretion of an assembled tetrameric CH2-deleted antibody in E. coli

Abstract

We have expressed in E. coli a functional assembled antibody variant that is secreted into the media. The antibody variant is a CH2-deleted chimeric antibody 14.18, which was previously shown to be a potentially useful reagent for radioimmunodetection of human tumors. The bacterial expression vector contains a dicistronic unit comprised of a L-chain cDNA and a CH2-deleted H-chain cDNA. For translocation across the bacterial membranes, we have replaced the natural signal peptides of the H and L chains with the signal peptide of the bacterial protein pectate lyase B. When expressed in the JM105 host under the control of the trp-lac promoter, the products were secreted into the M9 growth media to about 350 micrograms/L. The secreted antibody, which can be readily purified from the media without any denaturation or renaturation steps, retains antigen-binding activity. SDS-PAGE and nondenaturing size exclusion high-pressure liquid chromatography showed that it is a mixture of assembled HL and fully assembled H2L2. In H2L2, inter-H chain disulfide bonds are not formed, and the two HL half-molecules are likely held together by the trans interaction between the two CH3 domains.