title: Improved quantitative and qualitative production of single-domain intrabodies mediated by the co-expression of Erv1p sulfhydryl oxidase.
abstract: Camelidae single domain antibodies (VHHs) have structural and binding features that render them suitable alternatives to conventional IgG antibodies. VHHs are usually easier to produce as recombinant proteins than other antibody fragments. However, for some of the biotechnological applications for which they have been proposed, such as immunochromatography and assisted-crystallography, large amounts of purified antibodies are necessary, whereas some VHH-fusions with common tags such as GFP and SNAP are poorly expressed in the bacterial periplasm. Here we have shown that the co-expression of Erv1p sulfhydryl oxidase resulted in an astonishing yield increase of VHH-SNAP constructs expressed in the bacterial cytoplasm. The resulting recombinant antibodies were also more stable than the antibodies produced using the same plasmid, but in wild-type bacteria. Using this approach, it was possible to obtain tens of milligram of purified fusion antibodies using a basic flask fermentation protocol. Therefore, the described method represents a valid solution to produce inexpensively large amounts of single domain antibodies for in vitro applications and we expect it will be suitable for the production of other antibody fragments.
authors.affiliation: Protein Chemistry Unit, Cogentech, Milano, Italy.
authors.forename: Gianluca
authors.initials: G
authors.lastname: Veggiani
authors.forename: Ario
authors.initials: A
authors.lastname: de Marco
mesh_terms: D000818:Animals; D000906:Antibodies / Q000235:genetics* / Q000302:isolation & purification; D001709:Biotechnology; D002162:Camelus / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D010088:Oxidoreductases / Q000235:genetics* / Q000302:isolation & purification; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000235:genetics* / Q000302:isolation & purification; D050862:Oxidoreductases Acting on Sulfur Group Donors
chemical_list: D000906:Antibodies; D010088:Oxidoreductases; D011993:Recombinant Fusion Proteins; D050862:Oxidoreductases Acting on Sulfur Group Donors