Expression and secretion of an assembled tetrameric CH2-deleted antibody in E. coli.
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.
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Abbott Biotech, Inc., Needham Heights, MA.
Roy
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A
Foley
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SF
Coll
J T
JT
Gillies
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D000941:Antigens / Q000378:metabolism; D001483:Base Sequence; D001667:Binding, Competitive; D004247:DNA / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D005803:Genes, Immunoglobulin; D005822:Genetic Vectors; D006801:Humans; D007136:Immunoglobulins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics*; D008969:Molecular Sequence Data; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics
D000941:Antigens; D007136:Immunoglobulins; D011993:Recombinant Fusion Proteins; D004247:DNA