title: Functional solubilization of aggregation-prone HIV envelope proteins by covalent fusion with chaperone modules.
abstract: The envelope proteins of human immunodeficiency virus (HIV) and human T-cell lymphotrophic virus (HTLV) mediate cell attachment and membrane fusion. For HIV-1, the precursor protein gp160 is cleaved proteolytically into two fragments, the surface-associated receptor binding subunit gp120 and the membrane spanning subunit gp41, which is involved in membrane fusion during virus entry. Soluble and immunoreactive variants of gp41 are essential for the reliable diagnosis of HIV-1 infections. Hitherto, gp41 was solubilized by adding detergents, or in acidic or alkaline solvents. We find that covalent fusions with SlyD or FkpA, two homodimeric Escherichia coli chaperones with peptidyl-prolyl isomerase activity, solubilize retroviral envelope proteins without compromising their immunological reactivity. gp41 from HIV-1, gp36 from HIV-2 and gp21 from HTLV could be expressed in large amounts in the Escherichia coli cytosol when fused with one or two subunits of SlyD or FkpA. The fusion proteins could be easily isolated and refolded, and showed high solubility and immunoreactivity, thus providing sensitive and reliable tools for diagnostic applications. Covalent fusions with SlyD or FkpA might be valuable generic tools for the solubilization and activation of aggregation-prone proteins.
authors.affiliation: Roche Diagnostics GmbH, Nonnenwald 2, D-82377 Penzberg, Germany. christian.scholz@roche.com
authors.forename: Christian
authors.initials: C
authors.lastname: Scholz
authors.forename: Peter
authors.initials: P
authors.lastname: Schaarschmidt
authors.forename: Alfred Michael
authors.initials: AM
authors.lastname: Engel
authors.forename: Herbert
authors.initials: H
authors.lastname: Andres
authors.forename: Urban
authors.initials: U
authors.lastname: Schmitt
authors.forename: Elke
authors.initials: E
authors.lastname: Faatz
authors.forename: Jochen
authors.initials: J
authors.lastname: Balbach
authors.forename: Franz Xaver
authors.initials: FX
authors.lastname: Schmid
mesh_terms: D000595:Amino Acid Sequence; D002850:Chromatography, Gel; D002942:Circular Dichroism; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D015700:HIV Envelope Protein gp41 / Q000737:chemistry* / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D015497:HIV-1 / Q000737:chemistry* / Q000235:genetics; D020104:Immunophilins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D018832:Molecular Chaperones / Q000737:chemistry* / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D008969:Molecular Sequence Data; D019696:Peptidylprolyl Isomerase / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D011489:Protein Denaturation; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D016415:Sequence Alignment; D012995:Solubility; D013696:Temperature
chemical_list: D029968:Escherichia coli Proteins; D015700:HIV Envelope Protein gp41; D008565:Membrane Proteins; D018832:Molecular Chaperones; D011993:Recombinant Fusion Proteins; C085606:SlyD protein, E coli; D020104:Immunophilins; D019696:Peptidylprolyl Isomerase