In vivo biotinylated recombinant antibodies: construction, characterization, and application of a bifunctional Fab-BCCP fusion protein produced in Escherichia coli.
We describe a novel vector system suitable for the efficient preparation of in vivo biotinylated antibody Fab fragments in Escherichia coli. The previously described pGE20 vector used for the functional expression of truncated heavy (Fd) and light (L) chains of Fab into the bacterial culture medium was modified by inserting the C-terminal 101-amino-acid polypeptide of the biotin carboxyl carrier protein subunit of E. coli acetyl-CoA carboxylase (BCCP*). The secreted Fd-BCCP* fusion and L chain proteins were found to be disulfide linked and Fab-BCCP* complexes of an IgG1 antibody (Mab4) to human tumor necrosis factor alpha (TNF) were shown to retain both antigen and streptavidin-binding activities. The capacity of the Fab4 linked to BCCP* to bind TNF was identical to that observed with unmodified Fab4. Up to 15% of the expressed hybrids were able to interact with streptavidin when exogeneous d-biotin was added into the bacterial culture medium. The Fab4-BCCP* molecules were found to be more efficient than Fab4 linked to an engineered streptavidin-affinity tag for the detection of antigen on solid phase. In addition, we show here that the bacterially expressed Fab4-BCCP* complexes, adsorbed to streptavidin-agarose beads, can be used for the one-step purification of recombinant TNF by immunoaffinity chromatography.
Weiss
E
E
Ecole Supérieure de Biotechnologie de Strasbourg, Illkirch, France.
Chatellier
J
J
Orfanoudakis
G
G
D000103:Acetyl-CoA Carboxylase / Q000096:biosynthesis* / Q000235:genetics; D000469:Alkaline Phosphatase; D000595:Amino Acid Sequence; D000937:Antigen-Antibody Reactions; D001426:Bacterial Proteins; D001483:Base Sequence; D001710:Biotin; D002352:Carrier Proteins / Q000096:biosynthesis* / Q000235:genetics; D002846:Chromatography, Affinity / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D054889:Fatty Acid Synthase, Type II; D005822:Genetic Vectors; D007140:Immunoglobulin Fab Fragments / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics; D019809:Streptavidin; D014409:Tumor Necrosis Factor-alpha / Q000276:immunology / Q000302:isolation & purification*
D001426:Bacterial Proteins; D002352:Carrier Proteins; D007140:Immunoglobulin Fab Fragments; D011993:Recombinant Fusion Proteins; D014409:Tumor Necrosis Factor-alpha; D001710:Biotin; D019809:Streptavidin; D000469:Alkaline Phosphatase; D054889:Fatty Acid Synthase, Type II; D000103:Acetyl-CoA Carboxylase; C079633:biotin carboxyl carrier protein