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T‐Cell‐Targeted Immunofusion Proteins from E. coli
Author(s) -
BETTER MARC
Publication year - 1996
Publication title -
annals of the new york academy of sciences
Language(s) - Uncategorized
Resource type - Journals
SCImago Journal Rank - 1.712
H-Index - 248
eISSN - 1749-6632
pISSN - 0077-8923
DOI - 10.1111/j.1749-6632.1996.tb40592.x
Subject(s) - fusion protein , escherichia coli , antibody , ribosome inactivating protein , antigen , biochemistry , bacteria , biology , chemistry , microbiology and biotechnology , gene , recombinant dna , ribosome , rna , genetics , immunology
Antibodies and antibody domains are ideal agents for targeting the surface of cells, and fusion proteins between cell-targeting domains and cytotoxic proteins may be particularly effective therapeutic reagents. We constructed a family of immunofusion proteins linking the humanized Fab, F(ab')2, or single-chain antibody form of the H65 antibody (which recognizes the CD5 antigen on the surface of human T cells) with the plant ribosome-inactivating protein gelonin. To maximize the product yield and simplify the production process, each fusion protein was linked to a bacterial signal sequence for expression in E. coli as a secreted protein. More than 30 fusion genes were assembled with antibody domains and gelonin in various physical orientations. Each immunofusion accumulated in the bacterial culture supernatant in a properly folded, active form. Bacteria transformed with each fusion gene were then grown in a fermentor, and product was recovered from the cell-free fermentation broth by column chromatography. All of the immunofusion proteins were purified by a single process and each was tested for cytotoxicity toward antigen-positive human cells. A compact cGMP fermentation area was built to manufacture these fusion proteins. Our integrated approach to microbial protein production, including molecular genetics, bacterial fermentation, and initial isolation, is described in detail.