Novel strategy for the selection of human recombinant Fab fragments to membrane proteins from a phage-display library.
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-1IHI 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 beta-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-P1IHI, 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-P1IHI vesicles disrupted by Nonidet P-40 (NP-P1IHI). Both membrane-associated and soluble Ags were selected for vaccinia-specific clones (OG-P1IHI: 59/75 and NP-P1IHI: 1/75) and HIV-1-specific clones (OG-P1IHI: 11/75 and NP-P1IHI: 65/75) using our approach. Hence, the novel panning strategy described here may be applicable for selection of phage-libraries against membrane proteins.
Labrijn
Aran F
AF
Department of Immunopathology, CLB and Laboratory of Experimental and Clinical Immunology, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands. A_Labrijn@clb.nl
Koppelman
Marco H G M
MH
Verhagen
Janneke
J
Brouwer
Mieke C
MC
Schuitemaker
Hanneke
H
Hack
C Erik
CE
Huisman
Han G
HG
D000595:Amino Acid Sequence; D000818:Animals; D000941:Antigens / Q000302:isolation & purification; D001483:Base Sequence; D002460:Cell Line; D004247:DNA / Q000235:genetics; D016172:DNA Fingerprinting; D003902:Detergents; D015686:Gene Products, env / Q000276:immunology; D015483:HIV Antibodies / Q000235:genetics / Q000302:isolation & purification; D015488:HIV Antigens / Q000276:immunology; D006679:HIV Seropositivity / Q000235:genetics / Q000276:immunology; D015497:HIV-1 / Q000276:immunology; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000235:genetics* / Q000302:isolation & purification*; D008565:Membrane Proteins / Q000276:immunology* / Q000302:isolation & purification; D008969:Molecular Sequence Data; D019151:Peptide Library; D011817:Rabbits; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification; D012995:Solubility
D000941:Antigens; D003902:Detergents; D015686:Gene Products, env; D015483:HIV Antibodies; D015488:HIV Antigens; D007140:Immunoglobulin Fab Fragments; D008565:Membrane Proteins; D019151:Peptide Library; D011994:Recombinant Proteins; D004247:DNA