Multifunctional Inorganic-Binding Beads Self-Assembled Inside Engineered Bacteria

Abstract

Multifunctional shell-core nano/microbeads with a hydrophobic biopolymer core and a designed protein coat for selective binding of an inorganic substance and antibodies were self-assembled inside engineered bacteria. Hybrid genes were constructed to produce tailormade bead-coating proteins in the bacterium Escherichia coli. These fusion proteins contained a binding peptide for an inorganic material, the antibody binding ZZ domain, and a self-assembly promoting as well as biopolymer synthesizing enzyme. Production of these multidomain fusion proteins inside E. coli resulted in self-assembly of beads comprising a biopolyester core and displaying covalently bound binding sites for specific and selective binding of an inorganic substance and any antibody belonging to the immunoglobulin G class. Engineered beads were isolated and purified from the respective E. coli cells by standard cell disruption procedures. Bead morphology and the binding functionalities displayed at the bead surface were assessed by the enzyme-linked immunosorbent assay, transmission electron microscopy, elemental analysis, backscattering electron density, analytical density ultracentrifugation, and atomic force microscopy. These analyses showed that bacteria can be engineered to produce fusion proteins mediating self-assembly of spherical biopolymer beads with binding affinity to gold and/or silica and antibodies. Spherical structures of this type could conceivably serve as nano/microdevices for bioimaging in medical approaches where an antibody mediated targeted delivery of an inorganic contrast agent would be desired.

New Skin Test for Detection of Bovine Tuberculosis on the Basis of Antigen-Displaying Polyester Inclusions Produced by Recombinant Escherichia coli

“…After further washing, o-phenylenediamine (OPD) substrate (Abbott Diagnostics, IL, USA) was added and incubated for 15 min at room temperature. The reaction was stopped with 0.5 N H 2 SO 4 , and the absorbance was measured at 490 nm on an ELx808iu ultramicrotiter plate reader (Bio-Tek Instruments Inc., USA) (16). Results were expressed as optical density units at 490 nm.…”

“…Examples of foreign protein functions which have been successfully displayed on polyester beads using PhaC engineering are enzymes, binding domains/peptides, and antigens (13)(14)(15). Simultaneous fusions of several foreign proteins as well as coproduction of different PhaC fusion proteins enabled the codisplay of different protein functions on the same polyester bead with defined stoichiometry of the various proteins (12,(16)(17)(18). Foreign target proteins have been displayed on polyester beads for subsequent cleavage and purification (19)(20)(21)(22).…”

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).…”

Bioengineering of Bacterial Polymer Inclusions Catalyzing the Synthesis of N -Acetylneuraminic Acid

“…E. coli cell pellets containing polyester were resuspended in 50 mM potassium phosphate buffer (pH 7.5) and mechanically disrupted using a constant cell disruption system at 20 kpsi with a one-shot head adapter (Constant Systems Ltd., Northants, United Kingdom), and the lysate was subjected to centrifugation (4,000 ϫ g, 20 min, 4°C) to sediment the PHA beads. Final separation of the beads from the bacterial lysate was achieved by ultracentrifugation (150,000 ϫ g, 2 h, 10°C) on a glycerol gradient (22). Isolated PHA beads were stored at 4°C in 50 mM potassium phosphate buffer (pH 7.5) supplemented with 20% (vol/vol) ethanol until required.…”