Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.444203
Title: Production of HIV-2 envelope glycoproteins for structural and functional studies
Author: Halley, Jennifer Mary
ISNI:       0000 0001 3527 7470
Awarding Body: UCL (University College London)
Current Institution: University College London (University of London)
Date of Award: 2007
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Abstract:
Envelope glycoproteins of HIV-1 (gp160 > gp120/41) and HIV-2 (gp140 > gp105/36) have -40% sequence identity, bind cell surface receptors (CD4 and CCR5 or CXCR4) and effect membrane fusion, suggesting a close structural similarity. HIV-2 gp140 is more stable than HIV-1 gp160 (Sattentau et al., 1993), possibly making it a better candidate for structural studies. We have generated HIV-2 env-gene constructs that allow expression of soluble gp120 (gp105 with a truncated gp36 gp15 lacking the membrane anchor and cytoplasmic tail) in mammalian cells. HIV-2 env-genes have been rescued from the HIV-2ROd prototype and six HIV- infected individuals resident in Caio, Guinea Bissau. A range of constructs have been generated to enable efficient secretion of gp120s into tissue culture supernatant (TCSN) and/or enhance stability of the glycoproteins. These result in truncation at one of two positions in the gp36 coding region upstream of the membrane anchor, removal of the gp105/36 processing site and, introduction of a trimer-stabilising motif from Bacteriophage T4 fibritin (GYIPEAPRDGQAYVRKDG- EVWLLSTFL) at the C-terminus of gp15. All constructs were cloned to express a hexa-His tag at their C-termini to aid purification of the protein, but this proved inefficient. All constructs have been screened for expression competence in a transient system based on transfection of Human Embryonic Kidney 293T cells and western blot detection of gp120 in cell lysates and TCSN. Seven HIV-2ROd stable cell lines, based on Chinese Hamster Ovary Cells (CHO K1), have been selected which constitutively express and secrete gp120. The secreted material is recognised by a panel of mapped anti-gp105 monoclonal antibodies (MAbs) and partially purified gp120 is functionally active for human CD4-binding in ELISA-based assays, indicating that it is probably in a native state. Purification is based on lectin (GNA)-affinity chromatography followed by a MAb immunoaffinity column (ARP 3085, NIBSC) and gel filtration (Superose 6). This, together with dynamic light scattering (polydispersity -23.3) and circular dichroism (performed up to 85 C) suggests production of a correctly folded, oligomeric (probably trimeric) protein of a purity suitable for structural studies. Purified proteins are being used to set up further structural and functional experiments including crystallisation trials and will be used to generate MAbs which may assist crystallisation and be useful for HIV-2 strain typing assays.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.444203  DOI: Not available
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