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Production, purification, crystallization and preliminary X-ray diffraction analysis of the HIV-2-neutralizing V3 loop-specific Fab fragment 7C8
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
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2009 (English)In: Acta Crystallographica. Section F: Structural Biology and Crystallization Communications, ISSN 1744-3091, E-ISSN 1744-3091, Vol. 65, 705-708 p.Article in journal (Refereed) Published
Abstract [en]

7C8 is a mouse monoclonal antibody that is specific for the third hypervariable loop (V3 loop) of the human immunodeficiency virus type 2 (HIV-2) associated protein gp125. Fab fragments of 7C8 effectively neutralize HIV-2. 7C8 was expressed and purified from a hybridoma cell line in order to establish the molecular basis underlying the specificity of the 7C8 antibody for the V3 loop as well as the specific role of the elongated third complementarity-determining region of the heavy chain (CDRH3). The antibody was digested with papain and Fab fragments were purified using size-exclusion chromatography. Hanging-drop vapour-diffusion crystallization techniques were employed and the protein was crystallized in 50 mM ammonium sulfate, 100 mM Tris-HCl pH 8.5, 25%(w/v) PEG 8000 and 2.5%(w/v) PEG 400 at 275 K. The analysed crystals belonged to the rhombohedral space group P3(2)21, with unit-cell parameters a = b = 100.1, c = 196.8 angstrom, and diffracted to 2.7 angstrom resolution.

Place, publisher, year, edition, pages
2009. Vol. 65, 705-708 p.
National Category
Medical and Health Sciences
URN: urn:nbn:se:uu:diva-128405DOI: 10.1107/S1744309109020685ISI: 000267530000013OAI: oai:DiVA.org:uu-128405DiVA: diva2:331533
Available from: 2010-07-23 Created: 2010-07-20 Last updated: 2010-07-23Bibliographically approved

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Department of Cell and Molecular Biology
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Acta Crystallographica. Section F: Structural Biology and Crystallization Communications
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