Construction of CD4-based chimeric molecules by chemical cross-linking.
A simple method by which a soluble form of human CD4 (sT4) is chemically coupled to various carriers using a bifunctional reagent is described. The cross-linking of sT4 and carriers is accomplished with sulfosuccinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate (sulfo-SMCC) which creates a covalent bond between free NH2 and SH groups. If necessary, SH groups are introduced at the surface of the carriers using 2-iminothiolane. The method is simple, fast and efficient, and creates a thioether (S--C) bond which cannot be cleaved and thus gives stability to the construct in vivo. As an example of the applicability of this approach, sT4 was coupled to human serum albumin, a monoclonal antibody, and red blood cells. We show that for all of the sT4 conjugates, the cross-linking procedure conserved the sT4 reactivity for the gp120 and anti-CD4 monoclonal antibodies. Additionally, the sT4-Ig conjugate retained the binding specificity of the Ig portion and the cross-linking of sT4 to RBC proved to be very efficient and homogeneous. Altogether, this procedure allows the construction of chimeric molecules that cannot be obtained by genetic engineering and this may present many useful applications in the preparation of CD4-based anti-HIV drugs which could be rapidly constructed and screened.
Idziorek
T
T
Laboratoire de Biologie et Génétique des Infections Rétrovirales, Bat. CERVI, Hôpital de la Pitié, Paris, France.
Klatzmann
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D
D015704:CD4 Antigens / Q000737:chemistry* / Q000378:metabolism; D003432:Cross-Linking Reagents; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D004912:Erythrocytes / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D015151:Immunoblotting; D008301:Maleimides; D012709:Serum Albumin / Q000737:chemistry / Q000378:metabolism; D012995:Solubility
D015704:CD4 Antigens; D003432:Cross-Linking Reagents; D008301:Maleimides; D012709:Serum Albumin; C071675:sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate