In Vivo Production of scFv-Displaying Biopolymer Beads Using a Self-Assembly-Promoting Fusion Partner

• Published in: Bioconjugate chemistry 2008-01, Vol.19 (1), p.254-262
• Main Authors: Grage, Katrin, Rehm, Bernd H. A
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Antigens; beta-Galactosidase - immunology; beta-Galactosidase - metabolism; Binding sites; Biopolymers; Biopolymers - chemistry; Cupriavidus necator - enzymology; E coli; Electrophoresis, Polyacrylamide Gel; Enzymes; Escherichia coli; Immunoenzyme Techniques - methods; Immunoglobulin Variable Region - biosynthesis; Immunoglobulin Variable Region - immunology; Microspheres; Molecular Sequence Data; Mutation; Recombinant Fusion Proteins - metabolism; Studies; Temperature
• ISSN: 1043-1802; 1520-4812
• DOI: 10.1021/bc7003473

Recombinant production and, in particular, immobilization of antibody fragments onto carrier materials are of high interest with regard to diagnostic and therapeutic applications. In this study, the recombinant production of scFv-displaying biopolymer beads intracellularly in Escherichia coli was investigated. An anti-β-galactosidase scFv (single chain variable fragment of an antibody) was C-terminally tagged with the polymer-synthesizing enzyme PhaC from Cupriavidus necator by generating the respective hybrid gene. The functionality of the anti-β-galactosidase scFv-PhaC fusion protein was assessed by producing the respective soluble fusion protein in an Escherichia coli AMEF mutant strain. AMEF (antibody-mediated enzyme formation) strains contain an inactive mutant β-galactosidase, which can be activated by binding of an anti-β-galactosidase antibody. In vivo activation of AMEF β-galactosidase indicated that the scFv is functional with the C-terminal fusion partner PhaC. It was further demonstrated that polymer biosynthesis and bead formation were mediated by the scFv-PhaC fusion protein in the cytoplasm of recombinant E. coli when the polymer precursor was metabolically provided. This suggested that the C-terminal fusion partner PhaC acts as a functional insolubility partner, providing a natural cross-link to the bead and leading to in vivo immobilization of the scFv. Overproduction of the fusion protein at the polymer bead surface was confirmed by SDS−PAGE and MALDI-TOF/MS analysis of purified beads. Antigen binding functionality and specificity of the beads was assessed by analyzing the binding of β-galactosidase to scFv-displaying beads and subsequently eluting the bound protein at pH 2.7. A strong enrichment of β-galactosidase suggested the functional display of scFv at the bead surface as well as the applicability of these beads for antigen purification. Binding of β-galactosidase to the scFv-displaying beads was quantitatively analyzed by enzyme-linked assays measuring β-galactosidase activity. These indicated that the anti-β-galactosidase scFv-displaying beads bound a maximum of 38 ng of β-galactosidase per 1 µg of bead protein, showing an apparent equilibrium dissociation constant (K D) of 12 × 10−7 M. This study clearly demonstrated that anti-β-galactosidase scFv-displaying polymer beads can be produced in engineered E. coli in a one-step process by using PhaC as a self-assembly-promoting fusion partner.