Genetic Fusion of an Anti-BclA Single-Domain Antibody with Beta Galactosidase

Abstract

The Bacillus collagen-like protein of anthracis (BclA), found in Bacillus anthracis spores, is an attractive target for immunoassays. Previously, using phage display we had selected llama-derived single-domain antibodies that bound to B. anthracis spore proteins including BclA. Single-domain antibodies (sdAbs), the recombinantly expressed heavy domains from the unique heavy-chain-only antibodies found in camelids, provide stable and well-expressed binding elements with excellent affinity. In addition, sdAbs offer the important advantage that they can be tailored for specific applications through protein engineering. A fusion of a BclA targeting sdAb with the enzyme Beta galactosidase (β-gal) would enable highly sensitive immunoassays with no need for a secondary reagent. First, we evaluated five anti-BclA sdAbs, including four that had been previously identified but not characterized. Each was tested to determine its binding affinity, melting temperature, producibility, and ability to function as both capture and reporter in sandwich assays for BclA. The sdAb with the best combination of properties was constructed as a fusion with β-gal and shown to enable sensitive detection. This fusion has the potential to be incorporated into highly sensitive assays for the detection of anthrax spores.

Optimizing the Design of Diatom Biosilica-Targeted Fusion Proteins in Biosensor Construction for Bacillus anthracis Detection

“…Problems arise when combining peptides with incompatible fusion preferences, as was found to be the case in creating a series of diatom biosilica-targeted constructs encoding sdAbs against the B. anthracis S-layer protein EA1. In particular, both the Sil3 T8 (e.g., [12,15,16,32]) and sdAb EA1 (e.g., [33,34]) peptides previously have been made only as N-terminal fusions to their partners. One reason for this order of fusion partners is the requirement of many signal peptides' proximity to the protein terminus, rather than just their amino acid sequence.…”

Selection of Single-Domain Antibodies towards Western Equine Encephalitis Virus

“…Additionally, several strategies have been described for increasing the stability of sdAb [37], and have been shown to increase melting temperatures by as much as 20 °C [43]. Similarly, sdAb can be engineered to tailor them for specific assay formats [28,32,55].…”

Recombinant expression of nanobodies and nanobody-derived immunoreagents

“…The bacterial cytoplasm is a reducing environment that impairs the formation of the nanobody internal disulfide bond(s). However, several VHHs can fold into a functional structure independently on the presence of such disulfide bond(s) [49,102,103] and disulfide bond-independent nanobodies (intrabodies) were successfully produced in E. coli cytoplasm even when fused to large partners such cucurmosin toxin [104]. Since the preliminary results indicated that their anti-Bacilus anthracis nanobodies were stable and functional intrabodies, Anderson et al [103] expressed them fused to beta galactosidase directly in the cytoplasm of Tuner (DE3) E. coli.…”

“…However, several VHHs can fold into a functional structure independently on the presence of such disulfide bond(s) [49,102,103] and disulfide bond-independent nanobodies (intrabodies) were successfully produced in E. coli cytoplasm even when fused to large partners such cucurmosin toxin [104]. Since the preliminary results indicated that their anti-Bacilus anthracis nanobodies were stable and functional intrabodies, Anderson et al [103] expressed them fused to beta galactosidase directly in the cytoplasm of Tuner (DE3) E. coli. Shibuya et al [105] managed to coexpress in bacterial cytoplasm nanobodies fused to complementary split intein moieties for obtaining bispecific VHH constructs by in-cell transsplicing whereas nanobodies engineered with extra cysteines or ascorbate peroxidase were functionally recovered after production in NEB express F' cells [39].…”