title: High level production of functional antibody Fab fragments in an oxidizing bacterial cytoplasm.
abstract: The antigen-binding fragments (Fab) of antibodies are powerful tools in clinical therapy, molecular diagnostics and basic research. However, their principal applications require pure recombinant molecules in large amounts, which are challenging to obtain. Severe limitations in yield, folding and functionality are commonly encountered in bacterial production of Fab fragments. Secretion into the oxidizing periplasm generally results in low yield, whereas expression in the reducing cytoplasmic environment produces unfolded or non-functional protein. We hypothesized that an impaired reducing environment of the cytoplasm would permit correctly folded, functional cytoplasmic expression of Fabs with high yield. We used the Escherichia coli strain FA113, which has no activity of both thioredoxin and glutathione reductase, and thus has an oxidizing cytoplasmic environment. With the newly constructed vector pFAB1 we tested the cytoplasmic expression of two Fab fragments, which recognize the integral membrane protein NhaA, a bacterial Na(+)/H(+) antiporter. These antibodies differ in terms of DNA sequence and stability. Both antibody fragments were produced to very high yields (10-30 mg/l from bacterial cultures at an A(600 nm)=1.2-1.3). This is a factor 50-250 times higher than any other reported over-expression strategy for Fab fragments and currently represents the highest production rate ever been reported for antibody Fab fragments in bacteria grown to similar cell densities. The fragments are fully functional and can be efficiently purified by His-tag chromatography. Expression of active Fab fragments in the bacterial cytoplasm unlocks the possibility of using antibody specific targeting in an intracellular environment. Such a capacity opens new perspectives for investigating metabolic and regulatory pathways in vivo and also provides a powerful selection system for functional genomics.
authors.affiliation: Max-Planck-Institut für Biophysik, Abteilung für Molekulare Membranbiologie, Heinrich-Hoffmann-Str.7, Frankfurt am Main, D-60528, Germany.
authors.forename: Miro
authors.initials: M
authors.lastname: Venturi
authors.forename: Claudia
authors.initials: C
authors.lastname: Seifert
authors.forename: Carola
authors.initials: C
authors.lastname: Hunte
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D015153:Blotting, Western; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D003593:Cytoplasm / Q000737:chemistry / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000166:cytology / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins; D015870:Gene Expression; D005980:Glutathione Reductase / Q000235:genetics / Q000378:metabolism; D007140:Immunoglobulin Fab Fragments / Q000096:biosynthesis* / Q000737:chemistry / Q000276:immunology* / Q000302:isolation & purification; D007074:Immunoglobulin G / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D007135:Immunoglobulin Variable Region / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D007145:Immunoglobulin kappa-Chains / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D051379:Mice; D009154:Mutation / Q000235:genetics; D016877:Oxidants / Q000378:metabolism*; D010084:Oxidation-Reduction; D019897:Periplasm / Q000737:chemistry / Q000378:metabolism*; D011489:Protein Denaturation; D017510:Protein Folding; D017923:Sodium-Hydrogen Exchangers / Q000276:immunology; D013879:Thioredoxins / Q000235:genetics / Q000378:metabolism
chemical_list: D000911:Antibodies, Monoclonal; D029968:Escherichia coli Proteins; D007140:Immunoglobulin Fab Fragments; D007074:Immunoglobulin G; D007135:Immunoglobulin Variable Region; D007145:Immunoglobulin kappa-Chains; C102839:NhaA protein, E coli; D016877:Oxidants; D017923:Sodium-Hydrogen Exchangers; D013879:Thioredoxins; D005980:Glutathione Reductase