Engineering of <i>Escherichia coli</i> β‐galactosidase for solvent display of a functional scFv antibody fragment

Alcalá, Pilar; Ferrer-Miralles, Neus; Villaverde, Antonio

doi:10.1016/s0014-5793(02)03775-4

Cited by 5 publications

(5 citation statements)

References 33 publications

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“…Despite the considerable advantages of the E . coli system for recombinant protein production, there are major technical hurdles like the formation of inclusion bodies as a consequence of high-level protein production with low amounts of active protein contained in insoluble aggregates in the cytoplasm [32]. This study had three aims related to Pvs 48/45: First, to optimize its expression using full-length codon harmonization.…”

Section: Introductionmentioning

confidence: 99%

Recombinant Pvs48/45 Antigen Expressed in E. coli Generates Antibodies that Block Malaria Transmission in Anopheles albimanus Mosquitoes

et al. 2015

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Transmission of malaria parasites from humans to Anopheles mosquitoes can be inhibited by specific antibodies elicited during malaria infection, which target surface Plasmodium gametocyte/gamete proteins. Some of these proteins may have potential for vaccine development. Pvs48/45 is a P. vivax gametocyte surface antigen orthologous to Pfs48/45, which may play a role during parasite fertilization and thus has potential for transmission blocking (TB) activity. Here we describe the expression of a recombinant Pvs48/45 protein expressed in Escherichia coli as a ∼60kDa construct which we tested for antigenicity using human sera and for its immunogenicity and transmission blocking activity of specific anti-mouse and anti-monkey Pvs48/45 antibodies. The protein reacted with sera of individuals from malaria-endemic areas and in addition induced specific IgG antibody responses in BALB/c mice and Aotus l. griseimembra monkeys. Sera from both immunized animal species recognized native P. vivax protein in Western blot (WB) and immunofluorescence assays. Moreover, sera from immunized mice and monkeys produced significant inhibition of parasite transmission to An. Albimanus mosquitoes as shown by membrane feeding assays. Results indicate the presence of reactive epitopes in the Pvs48/45 recombinant product that induce antibodies with TB activity. Further testing of this protein is ongoing to determine its vaccine potential.

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Section: Introductionmentioning

confidence: 99%

Recombinant Pvs48/45 Antigen Expressed in E. coli Generates Antibodies that Block Malaria Transmission in Anopheles albimanus Mosquitoes

et al. 2015

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“…When examining the combination of properties (cytoplasmic expression, affinity, melting temperature, and function in immunoassays for the detection of BclA), clone A5 was the best choice for construction of a β-gal fusion. Previously, researchers demonstrated that β-gal was tolerant to N-terminal fusions and could be produced genetically fused to a scFv for use in immunoassays [29]. We have expanded upon this prior work, demonstrating a functional genetic fusion between β-gal and an sdAb.…”

Section: Discussionmentioning

confidence: 92%

“…Additionally, β-gal is a tetramer with a molecular weight of 464 kDa, so fusions with this enzyme would also benefit from avidity. Previously, it had been reported that the enzyme β-gal is able to function with a scFv (linked variable heavy and variable light chain from a conventional antibody) inserted at the N-terminus of the enzyme [29]. Unlike fusions with AP, the β-gal fusions need to be produced in the cytoplasm.…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2018

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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.

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“…As the recognition module of an antibody, the variable region (Fv) presents a stable scaffold composed of structurally conserved framework regions and highly divergent complementarity-determining regions. To date, many Fvs have been the target of protein engineering and are fused with other molecules or proteins to create novel molecular probes, − sensor proteins, − and fusion enzymes for prodrug therapy. − …”

mentioning

confidence: 74%

Creation of a Ligand-Dependent Enzyme by Fusing Circularly Permuted Antibody Variable Region Domains

et al. 2016

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Allosteric control of enzyme activity with exogenous substances has been hard to achieve, especially using antibody domains that potentially allow control by any antigens of choice. Here, in order to attain this goal, we developed a novel antibody variable region format introduced with circular permutations, called Clampbody. The two variable-region domains of the antibone Gla protein (BGP) antibody were each circularly permutated to have novel termini at the loops near their domain interface. Through their attachment to the N- and C-termini of a circularly permutated TEM-1 β-lactamase (cpBLA), we created a molecular switch that responds to the antigen peptide. The fusion protein specifically recognized the antigen, and in the presence of some detergent or denaturant, its catalytic activity was enhanced up to 4.7-fold in an antigen-dependent manner, due to increased resistance to these reagents. Hence, Clampbody will be a powerful tool for the allosteric regulation of enzyme and other protein activities and especially useful to design robust biosensors.

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Engineering of Escherichia coli β‐galactosidase for solvent display of a functional scFv antibody fragment

Cited by 5 publications

References 33 publications

Recombinant Pvs48/45 Antigen Expressed in E. coli Generates Antibodies that Block Malaria Transmission in Anopheles albimanus Mosquitoes

Recombinant Pvs48/45 Antigen Expressed in E. coli Generates Antibodies that Block Malaria Transmission in Anopheles albimanus Mosquitoes

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

Creation of a Ligand-Dependent Enzyme by Fusing Circularly Permuted Antibody Variable Region Domains

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