title: Efficient expression of full-length antibodies in the cytoplasm of engineered bacteria.
abstract: Current methods for producing immunoglobulin G (IgG) antibodies in engineered cells often require refolding steps or secretion across one or more biological membranes. Here, we describe a robust expression platform for biosynthesis of full-length IgG antibodies in the Escherichia coli cytoplasm. Synthetic heavy and light chains, both lacking canonical export signals, are expressed in specially engineered E. coli strains that permit formation of stable disulfide bonds within the cytoplasm. IgGs with clinically relevant antigen- and effector-binding activities are readily produced in the E. coli cytoplasm by grafting antigen-specific variable heavy and light domains into a cytoplasmically stable framework and remodelling the fragment crystallizable domain with amino-acid substitutions that promote binding to Fcγ receptors. The resulting cytoplasmic IgGs—named 'cyclonals'—effectively bypass the potentially rate-limiting steps of membrane translocation and glycosylation.
authors: School of Chemical and Biomolecular Engineering, Cornell University, Ithaca, New York 14853, USA.
authors: Michael-Paul
authors: MP
authors: Robinson
authors: New England Biolabs, 240 County Road, Ipswich, Massachusetts 01938, USA.
authors: Na
authors: N
authors: Ke
authors: New England Biolabs, 240 County Road, Ipswich, Massachusetts 01938, USA.
authors: Julie
authors: J
authors: Lobstein
authors: School of Chemical and Biomolecular Engineering, Cornell University, Ithaca, New York 14853, USA.
authors: Cristen
authors: C
authors: Peterson
authors: School of Chemical and Biomolecular Engineering, Cornell University, Ithaca, New York 14853, USA.
authors: Alana
authors: A
authors: Szkodny
authors: School of Chemical and Biomolecular Engineering, Cornell University, Ithaca, New York 14853, USA.
authors: Thomas J
authors: TJ
authors: Mansell
authors: New England Biolabs, 240 County Road, Ipswich, Massachusetts 01938, USA.
authors: Corinna
authors: C
authors: Tuckey
authors: New England Biolabs, 240 County Road, Ipswich, Massachusetts 01938, USA.
authors: Paul D
authors: PD
authors: Riggs
authors: New England Biolabs, 240 County Road, Ipswich, Massachusetts 01938, USA.
authors: Paul A
authors: PA
authors: Colussi
authors: New England Biolabs, 240 County Road, Ipswich, Massachusetts 01938, USA.
authors: Christopher J
authors: CJ
authors: Noren
authors: New England Biolabs, 240 County Road, Ipswich, Massachusetts 01938, USA.
authors: Christopher H
authors: CH
authors: Taron
authors: School of Chemical and Biomolecular Engineering, Cornell University, Ithaca, New York 14853, USA.
authors: Matthew P
authors: MP
authors: DeLisa
authors: New England Biolabs, 240 County Road, Ipswich, Massachusetts 01938, USA.
authors: Mehmet
authors: M
authors: Berkmen
mesh_terms: D000906:Antibodies; D000917:Antibody Formation / Q000235:genetics*; D001435:Bacteriophages / Q000235:genetics; D015153:Blotting, Western; D003593:Cytoplasm / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000235:genetics*; D006031:Glycosylation; D007074:Immunoglobulin G / Q000096:biosynthesis*; D030781:Organisms, Genetically Modified / Q000235:genetics*; D010957:Plasmids / Q000235:genetics; D021381:Protein Transport; D020349:Surface Plasmon Resonance
chemical_list: D000906:Antibodies; D007074:Immunoglobulin G