Reconciling the Structural Attributes of Avian Antibodies

Conroy, Paul J.; Law, Ruby H. P.; Gilgunn, Sarah; Hearty, Stephen; Caradoc-Davies, Tom T.; Lloyd, Gordon J.; O’Kennedy, Richard; Whisstock, James C.

doi:10.1074/jbc.m114.562470

Cited by 27 publications

(21 citation statements)

References 54 publications

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“…Purification of both the C2 quad(410 -535) and C2 quad (410 -526) mutants was performed by periplasmic protein extraction with osmotic shock (19). The cell pellets were resuspended in binding/resuspension buffer (50 mM Tris/Cl (pH 8.0), 300 mM NaCl, 20 mM imidazole, and 0.02% NaN 3 (typical volume is 5 ml per 1 g of wet cells)).…”

Section: Methodsmentioning

confidence: 99%

Structural Basis for Ca2+-mediated Interaction of the Perforin C2 Domain with Lipid Membranes

Yagi

Conroy

Leung

et al. 2015

Journal of Biological Chemistry

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Background: Perforin is a critical component of immune homeostasis, responsible for clearing virally infected cells. Results: The molecular details of calcium binding by the perforin C2 domain are revealed. Conclusion: Calcium-mediated structural rearrangement activates perforin for membrane binding. Significance: The C2 domain regulates membrane binding by calcium-dependent events, which have now been defined for a mammalian perforin C2.

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Section: Methodsmentioning

confidence: 99%

Structural Basis for Ca2+-mediated Interaction of the Perforin C2 Domain with Lipid Membranes

Yagi

Conroy

Leung

et al. 2015

Journal of Biological Chemistry

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“…Antibodies are at the forefront of the field of targeted therapeutics and diagnostics due to their natural high affinity and excellent half-life properties [ 1 ]. These molecules can be readily manipulated using standard molecular biology techniques into specialised antibodies that are tailored to perform efficiently in their chosen end-point application [ 2 ]. The biopharmaceutical industry has heavily invested in antibody-based therapeutics, which currently represents the largest and fastest growing class of biopharmaceuticals [ 3 ].…”

Section: Introductionmentioning

confidence: 99%

Comprehensive N-Glycan Profiling of Avian Immunoglobulin Y

et al. 2016

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Recent exploitation of the avian immune system has highlighted its suitability for the generation of high-quality, high-affinity antibodies to a wide range of antigens for a number of therapeutic and biotechnological applications. The glycosylation profile of potential immunoglobulin therapeutics is species specific and is heavily influenced by the cell-line/culture conditions used for production. Hence, knowledge of the carbohydrate moieties present on immunoglobulins is essential as certain glycan structures can adversely impact their physicochemical and biological properties. This study describes the detailed N-glycan profile of IgY polyclonal antibodies from the serum of leghorn chickens using a fully quantitative high-throughput N-glycan analysis approach, based on ultra-performance liquid chromatography (UPLC) separation of released glycans. Structural assignments revealed serum IgY to contain complex bi-, tri- and tetra-antennary glycans with or without core fucose and bisects, hybrid and high mannose glycans. High sialic acid content was also observed, with the presence of rare sialic acid structures, likely polysialic acids. It is concluded that IgY is heavily decorated with complex glycans; however, no known non-human or immunogenic glycans were identified. Thus, IgY is a potentially promising candidate for immunoglobulin-based therapies for the treatment of various infectious diseases.

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“…Application of different methodologies available for antibody purifi cation is widespread, as underlined in some thematic papers, e.g. [33][34][35]. Protein L agarose purifi cation is a one-step Additionally, the purifi cation results were confi rmed by Western blot analysis (Figure 3).…”

Section: Resultsmentioning

confidence: 99%

Different Strategies used in the Production of human monoclonal scfv Antibodies Specific to Dimers of Membrane Receptors

Łukasiewicz¹

2017

J Biol Med

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Phage display technology is one of the methods widely adopted for the production of human monoclonal antibodies. The subject of the present research was the scFv -single chain variable fragment antibody -which was able to recognize the D 2 -5-HT 1A heteromer formed by dopamine D 2 and serotonin 5-HT 1A receptors. The antibody was selected using the phage display technique and the human antibody scFv phagemid library Tomlinson J. We investigated two expression systems: prokaryotic and eukaryotic. The results indicate that both of these systems (E.coli HB2151 and High Five cells (BTI-TN-5B1-4) of Trichopulsia ni) are suitable for the production of scFv antibodies, but the scFvs obtained from insect cells could attain a higher protein concentration. Next, we examined a few purifi cation methods: immobilized-metal affi nity chromatography (IMAC), affi nity chromatography with the c-myc tag and the methods using protein L or A agarose, which are applicable to scFvs lacking tags. In the bacterial as well as insect cell expression systems, protein L agarose chromatography gave satisfactory results, and in our opinion, it is the best choice as a purifi cation resin. Additionally, circular dichroism measurements confi rmed the predominant content of secondary structures in the tested antibodies, which is consistent with the literature data.

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Reconciling the Structural Attributes of Avian Antibodies

Cited by 27 publications

References 54 publications

Structural Basis for Ca2+-mediated Interaction of the Perforin C2 Domain with Lipid Membranes

Structural Basis for Ca2+-mediated Interaction of the Perforin C2 Domain with Lipid Membranes

Comprehensive N-Glycan Profiling of Avian Immunoglobulin Y

Different Strategies used in the Production of human monoclonal scfv Antibodies Specific to Dimers of Membrane Receptors

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