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

Abstract

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-beta-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-beta-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 beta-galactosidase, which can be activated by binding of an anti-beta-galactosidase antibody. In vivo activation of AMEF beta-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 beta-galactosidase to scFv-displaying beads and subsequently eluting the bound protein at pH 2.7. A strong enrichment of beta-galactosidase suggested the functional display of scFv at the bead surface as well as the applicability of these beads for antigen purification. Binding of beta-galactosidase to the scFv-displaying beads was quantitatively analyzed by enzyme-linked assays measuring beta-galactosidase activity. These indicated that the anti-beta-galactosidase scFv-displaying beads bound a maximum of 38 ng of beta-galactosidase per 1 microg of bead protein, showing an apparent equilibrium dissociation constant ( KD) of 12 x 10 (-7) M. This study clearly demonstrated that anti-beta-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.

A novel N-terminal region of the membrane β-hexosyltransferase: its role in secretion of soluble protein by Pichia pastoris

“…As such, scFv13R4 has been heterologously produced in E. coli, Sac. cerevisiae and Chinese hamster ovary cells (Visintin et al, 1999;Grage & Rehm, 2008;Bach et al, 2001). The BHT containing the classical leader followed by putative non-classical signal, the BHT enclosing the putative classical leader, the BHT enclosing the putative non-classical signal and the MFa leader were placed in-frame at the amino-terminal position followed by scFv13R4.…”

One-Step Production of Immobilized α-Amylase in Recombinant Escherichia coli

“…In order to identify the BLA-PhaC fusion protein, the band of interest was cut out from the gel and subjected to matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). Peptides produced by tryptic digestion were analyzed as previously described (6).…”

Bacterial Polyester Inclusions Engineered To Display Vaccine Candidate Antigens for Use as a Novel Class of Safe and Efficient Vaccine Delivery Agents

“…Beads have been developed with covalently attached enzymes, suggesting an application in immobilization and stabilization of biocatalysts (22). Recently, biopolyester beads have been produced which display immobilized antibody single-chain fragments as well as multiple binding functions, including the binding of inorganic compounds (4,11,14).…”