Antibody fusions with fluorescent proteins: a versatile reagent for profiling protein expression

Morino, Kazuhide; Katsumi, Harue; Akahori, Yasushi; Iba, Yoshitaka; Shinohara, Midori; Ukai, Yojiro; Kohara, Yuji; Kurosawa, Yoshikazu

doi:10.1016/s0022-1759(01)00462-8

Cited by 61 publications

(40 citation statements)

References 36 publications

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“…Colonies were picked up, and E. coli cells harboring phagemid were grown in 2ϫYT medium containing 100 g of ampicillin/ ml, 0.05% glucose, and 1 mM IPTG (isopropyl-␤-D-thiogalactopyranoside) at 30°C overnight. The Fab-cp3 form of MAb was secreted into medium (27). Culture supernatants containing Fab-cp3 molecules were subjected to enzyme-linked immunosorbent assay (ELISA) against 12 H3N2 strains and an H1N1 NC99 strain of influenza viruses.…”

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confidence: 99%

Conserved Neutralizing Epitope at Globular Head of Hemagglutinin in H3N2 Influenza Viruses

Iba

Fujii

Ohshima

et al. 2014

J Virol

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Neutralizing antibodies that target the hemagglutinin of influenza virus either inhibit binding of hemagglutinin to cellular receptors or prevent the low-pH-induced conformational change in hemagglutinin required for membrane fusion. In general, the former type of antibody binds to the globular head formed by HA1 and has narrow strain specificity, while the latter type binds to the stem mainly formed by HA2 and has broad strain specificity. In the present study, we analyzed the epitope and function of a broadly neutralizing human antibody against H3N2 viruses, F005-126. The crystal structure of F005-126 Fab in complex with hemagglutinin revealed that the antibody binds to the globular head, spans a cleft formed by two hemagglutinin monomers in a hemagglutinin trimer, and cross-links them. It recognizes two peptide portions (sites L and R) and a glycan linked to asparagine at residue 285 using three complementarity-determining regions and framework 3 in the heavy chain. Binding of the antibody to sites L (residues 171 to 173, 239, and 240) and R (residues 91, 92, 270 to 273, 284, and 285) is mediated mainly by van der Waals contacts with the main chains of the peptides in these sites and secondarily by hydrogen bonds with a few side chains of conserved sequences in HA1. Furthermore, the glycan recognized by F005-126 is conserved among H3N2 viruses. F005-126 has the ability to prevent low-pH-induced conformational changes in hemagglutinin. The newly identified conserved epitope, including the glycan, should be immunogenic in humans and may induce production of broadly neutralizing antibodies against H3 viruses. IMPORTANCEAntibodies play an important role in protection against influenza virus, and hemagglutinin is the major target for virus neutralizing antibodies. It has long been believed that all effective neutralizing antibodies bind to the surrounding regions of the sialic acid-binding pocket and inhibit the binding of hemagglutinin to the cellular receptor. Since mutations are readily introduced into such epitopes, this type of antibody shows narrow strain specificity. Recently, however, broadly neutralizing antibodies have been isolated. Most of these bind either to conserved sites in the stem region or to the sialic acid-binding pocket itself. In the present study, we identified a new neutralizing epitope in the head region recognized by a broadly neutralizing human antibody against H3N2. This epitope may be useful for design of vaccines.

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confidence: 99%

Conserved Neutralizing Epitope at Globular Head of Hemagglutinin in H3N2 Influenza Viruses

Iba

Fujii

Ohshima

et al. 2014

J Virol

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“…Colonies were picked up, and the E. coli colonies harboring the phagemid were grown in 2ϫ yeast extract-tryptophan (YT) medium containing 100 g/ml ampicillin, 0.05% glucose, and 1 mM isopropyl-␤-D-thiogalactopyranoside at 30°C overnight. The Fab-cp3 form of Ab was secreted into the medium (13). The culture supernatants containing Fab-cp3 molecules were subjected to enzymelinked immunosorbent assay (ELISA) against various H3 strains of influenza viruses and an H1 strain of influenza virus.…”

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confidence: 99%

Naturally Occurring Antibodies in Humans Can Neutralize a Variety of Influenza Virus Strains, Including H3, H1, H2, and H5

Ohshima

Iba

Kubota‐Koketsu

et al. 2011

J Virol

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114

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Influenza A viruses are classified into 16 subtypes according to the serotypes of hemagglutinin (HA). It is generally thought that neutralizing antibodies (Abs) are not broadly cross-reactive among HA subtypes. We examined the repertoire of neutralizing Abs against influenza viruses in humans. B lymphocytes were collected from donors by apheresis, and Ab libraries were constructed by using phage-display technology. Anti-HA clones were isolated by screening with H3N2 viruses. Their binding activity was examined, and four kinds of Abs showing broad strain specificity were identified from one donor. Two of the Abs, F045-092 and F026-427, were extensively analyzed. They neutralized not only H3N2 but also H1N1, H2N2, and H5N1 viruses, although the activities were largely varied. Flow cytometry suggested that they have the ability to bind to HA and HA1 artificially expressed on the cell surface. They show hemagglutination inhibition activity and do not compete with C179, an Ab thought to bind to the stalk region. F045-092 competes with Abs that recognize sites A and B for binding to HA. Furthermore, the serine at residue 136 in site A could be a part of the epitope. Thus, it is likely that F045-092 and F026-427 bind to a conserved epitope in the head region formed by HA1. Interestingly, while the V H 1-69 gene can encode MAbs against the HA stem that are group 1 specific, F045-092 and its relatives that recognize the head region also use V H 1-69. The possible epitope recognized by these clones is discussed.

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“…In addition, scFvs are typically used for antibody selection and engineering, in which many scFvs (10 to hundreds) often are simultaneously isolated (7, 11). Thus, fusion to GFP can enable rapid assessment of scFv binding and specificity properties without the requirement for chemical modification and the difficulties associated with losses in activity (2,8,18,20,21,29).The key to an effective scFv-GFP fusion protein platform is the ability to rapidly take an identified scFv or collection of scFvs and produce them efficiently as GFP fusion proteins in an appropriate host. Expression of scFv-GFP fusion proteins using bacteria has resulted in limited success, with yields of 100 to 200 g/liter, whether periplasmic secretion or inclusion body methods were used (2,8,18,20,21).…”

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A Yeast Platform for the Production of Single-Chain Antibody-Green Fluorescent Protein Fusions

Huang

Shusta

2006

Appl Environ Microbiol

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Fusion proteins comprised of a binding domain and green fluorescent protein (GFP) have the potential to act as one-step binding reagents. In this study, eight single-chain antibodies (scFv) and one single-chain T-cell receptor (scTCR) were secreted as fusions to GFP using a Saccharomyces cerevisiae expression system. Fusion protein secretion levels ranged over 3 orders of magnitude, from 4 g/liter to 4 mg/liter, and correlated well with the secretion levels of the unfused scFv/scTCR. Three fusion types with various linker lengths and fusion orientations were tested for each scFv/scTCR. Although the fusion protein secretion levels were not significantly affected by the nature of the fusion construct, the properties of the fusion protein were clearly influenced. The fluorescence yield per fusion molecule was increased by separating the scFv/scTCR and GFP with an extended (GGGGS) 3 linker, and fusions with scFv/scTCR at the carboxy-terminus were more resistant to degradation. By evaluating leader sequence processing and using GFP fluorescence to track intracellular processing, it was determined that the majority of fusion protein synthesized by the yeast was not secreted and in most cases was accumulating in an immature, although active, endoplasmic-reticulum (ER)-processed form. This contrasted with unfused scFv, which accumulated in both immature ER-processed and mature post-Golgi forms. The results indicated that yeast can be used as an effective host for the secretion of scFv/scTCR-GFP fusion proteins and that as a result of intracellular secretory bottlenecks, there is considerable yeast secretory capacity remaining to be exploited.Antibodies are widely used for cell immunostaining, flow cytometry, and cellular localization studies. Although many applications make use of a fluorescently labeled secondary antibody for detection purposes, direct conjugation of antibodies with organic fluorophores can also be used for visualization and quantification. Often, the conjugation of the antibody and fluorophore is performed using primary amine-coupling chemistry that results in the attachment of the fluorophore to lysine residues. However, lysine is frequently found within the antigen binding region, and fluorophore coupling can diminish or completely inhibit antigen binding activity (13,26). In addition, the environmental sensitivities of organic dyes and their susceptibilities to photobleaching are quite variable. Direct fusion of an antibody to green fluorescent protein (GFP) could overcome these disadvantages. Such a one-step immunoreagent would benefit from the fact that GFP is quite stable over a wide range of pH, temperature, and detergent conditions and is resistant to photobleaching (23). In terms of the antibody subunit of a GFP fusion protein, single-chain antibodies (scFvs) consisting of the variable heavy-and light-chain regions of intact antibodies retain binding activity and can be efficiently processed by microbial production hosts. In addition, scFvs are typically used for antibody selection and engine...

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Antibody fusions with fluorescent proteins: a versatile reagent for profiling protein expression

Cited by 61 publications

References 36 publications

Conserved Neutralizing Epitope at Globular Head of Hemagglutinin in H3N2 Influenza Viruses

Conserved Neutralizing Epitope at Globular Head of Hemagglutinin in H3N2 Influenza Viruses

Naturally Occurring Antibodies in Humans Can Neutralize a Variety of Influenza Virus Strains, Including H3, H1, H2, and H5

A Yeast Platform for the Production of Single-Chain Antibody-Green Fluorescent Protein Fusions

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