Novel strategy for the selection of human recombinant Fab fragments to membrane proteins from a phage-display library

• Published in: Journal of immunological methods 2002-03, Vol.261 (1), p.37-48
• Main Authors: Labrijn, Aran F, Koppelman, Marco H.G.M, Verhagen, Janneke, Brouwer, Mieke C, Schuitemaker, Hanneke, Hack, C.Erik, Huisman, Han G
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Animals; Antigens - isolation & purification; Base Sequence; Biological and medical sciences; Biotechnology; Cell Line; Detergents; DNA - genetics; DNA Fingerprinting; Fundamental and applied biological sciences. Psychology; Fundamental immunology; Galanthus nivalis; Gene Products, env - immunology; Genetic engineering; Genetic technics; HIV Antibodies - genetics; HIV Antibodies - isolation & purification; HIV Antigens - immunology; HIV Seropositivity - genetics; HIV Seropositivity - immunology; HIV-1; HIV-1 - immunology; Human immunodeficiency virus 1; Humans; Immunoglobulin Fab Fragments - genetics; Immunoglobulin Fab Fragments - isolation & purification; Membrane proteins; Membrane Proteins - immunology; Membrane Proteins - isolation & purification; Methods. Procedures. Technologies; Microbiology; Miscellaneous; Molecular immunology; Molecular Sequence Data; n-octyl ^b-d-glucopyranoside; n-Octyl β- d-glucopyranoside; Peptide Library; Phage-display libraries; Rabbits; Recombinant Fab; Recombinant Proteins - genetics; Recombinant Proteins - isolation & purification; Selection; Solubility; Synthetic digonucleotides and genes. Sequencing; Techniques
• ISSN: 0022-1759; 1872-7905
• DOI: 10.1016/S0022-1759(01)00542-7

Traditionally, the selection of phage-display libraries is performed on purified antigens (Ags), immobilized to a solid substrate. However, this approach may not be applicable for some Ags, such as membrane proteins, which for structural integrity strongly rely on their native environment. Here we describe an approach for the selection of phage-libraries against membrane proteins. The envelope glycoproteins (Env) of the Human Immunodeficiency Virus type-1 (HIV-1) were used as a model for a type-1 integral membrane protein. HIV-1 IHI Env, expressed on the surface of Rabbit Kidney cells (RK13) with a recombinant vaccinia virus (rVV), was solubilized using the non-ionic detergent n-Octyl β- d-glucopyranoside (OG). Membrane associated Env was reconstituted into vesicles by the simultaneous removal of detergent and free monomeric Env subunits by gel-filtration. The resulting antigen preparation, termed OG-P1 IHI, was captured on microtiter plates coated with Galanthus nivalis agglutinin (GNA) and used for rounds of selection (panning) of a well-characterized phage-display library derived from an HIV-1 seropositive donor. Simultaneously, an identical experiment was performed with OG-P1 IHI vesicles disrupted by Nonidet P-40 (NP-P1 IHI). Both membrane-associated and soluble Ags were selected for vaccinia-specific clones (OG-P1 IHI: 59/75 and NP-P1 IHI: 1/75) and HIV-1-specific clones (OG-P1 IHI: 11/75 and NP-P1 IHI: 65/75) using our approach. Hence, the novel panning strategy described here may be applicable for selection of phage-libraries against membrane proteins.