Live long and active: Polypeptide-mediated assembly of antibody variable fragments.
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ABSTRACT:
SUBMITTER: Lee C
PROVIDER: S-EPMC8382037 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Although combinatorial antibody libraries have solved the problem of access to large immunological repertoires, efficient production of these complex molecules remains a problem. Here we demonstrate the efficient expression of a unique large single-chain (lsc) antibody in the chloroplast of the unicellular, green alga, Chlamydomonas reinhardtii. We achieved high levels of protein accumulation by synthesizing the lsc gene in chloroplast codon bias and by driving expression of the chimeric gene using either of two C. reinhardtii chloroplast promoters and 5' and 3' RNA elements. This lsc antibody, directed against glycoprotein D of the herpes simplex virus, is produced in a soluble form by the alga and assembles into higher order complexes in vivo. Aside from dimerization by disulfide bond formation, the antibody undergoes no detectable posttranslational modification. We further demonstrate that accumulation of the antibody can be modulated by the specific growth regime used to culture the alga, and by the choice of 5' and 3' elements used to drive expression of the antibody gene. These results demonstrate the utility of alga as an expression platform for recombinant proteins, and describe a new type of single chain antibody containing the entire heavy chain protein, including the Fc domain.
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Project description:Potyviruses are major pathogens that often cause mixed infection in calla lilies. To reduce the time and cost of virus indexing, a detection method for the simultaneous targeting of multiple potyviruses was developed by generating a broad-spectrum monoclonal antibody (MAb) for detecting the greatest possible number of potyviruses. The conserved 121-amino-acid core regions of the capsid proteins of Dasheen mosaic potyvirus (DsMV), Konjak mosaic potyvirus (KoMV), and Zantedeschia mild mosaic potyvirus (ZaMMV) were sequentially concatenated and expressed as a recombinant protein for immunization. After hybridoma cell fusion and selection, one stable cell line that secreted a group-specific antibody, named C4 MAb, was selected. In the reaction spectrum test, the C4 MAb detected at least 14 potyviruses by indirect enzyme-linked immunosorbent assay (I-ELISA) and Western blot analysis. Furthermore, the variable regions of the heavy (VH) and light (VL) chains of the C4 MAb were separately cloned and constructed as single-chain variable fragments (scFvs) for expression in Escherichia coli. Moreover, the pectate lyase E (PelE) signal peptide of Erwinia chrysanthemi S3-1 was added to promote the secretion of C4 scFvs into the medium. According to Western blot analysis and I-ELISA, the soluble C4 scFv (VL-VH) fragment showed a binding specificity similar to that of the C4 MAb. Our results demonstrate that a recombinant protein derived from fusion of the conserved regions of viral proteins has the potential to produce a broad-spectrum MAb against a large group of viruses and that the PelE signal peptide can improve the secretion of scFvs in E. coli.
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Project description:Single-chain variable antibody fragments (scFvs) with a 2-amino-acid linker capable of multimerization as di-, tri-, or tetrabodies that neutralize bovine herpesvirus type 1 (BoHV-1) in vitro were constructed and expressed in Pichia pastoris. In contrast to the monomeric form, multimeric scFvs had a higher virus neutralization potency, as evidenced by a 2-fold increase in their ability to neutralize BoHV-1 due to avidity effects. Mass spectrum (quadrupole time of flight [Q-TOF]) analyses of multimeric scFv demonstrated extensive heterogeneity due to differential cleavage, variable glycosylation (1 to 9 mannose residues), and the incorporation of minor unidentified adducts. Regardless of the differential glycosylation patterns, the scFvs recognized non-gB or -gE target viral epitopes in the BoHV-1 envelope fraction in a Western blot and also neutralized BoHV-1 in infected Madin-Darby kidney (MDBK) cells in vitro. Indirect evidence for the noncovalent multimerization of scFv was the presence of a major peak of multimerized scFv without a His tag (due to differential cleavage) in the Q-TOF profile, unlike monomeric scFv, which copurified with normally His-tagged scFv and recognized the target antigen. Overall, differentially glycosylated recombinant scFvs against BoHV-1 with a short linker (2 amino acids) are capable of assembly into functional multimers that confer high avidity, resulting in increased virus neutralization in vitro compared to that of monovalent scFv with a long (18-amino-acid) flexible linker. Overall, recombinant multimerized scFv5-2L potentially provides a high-potency therapeutic and immunodiagnostic reagent against BoHV-1, which is suitable for passive immunization and topical application.
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