Rapid covalent coupling of proteins to cell surfaces: immunological characterization of viable protein-cell conjugates.
A rapid and efficient 2-step procedure is described for covalently attaching proteins to cell surfaces by using a heterobifunctional cross-linking agent, succinimidyl-4-(N-maleimidomethylcyclohexane)-1-carboxylate (SMCC). In the first step, protein is derivatized for about 30 min with a 1:1 (mole:mole) stoichiometric ratio of SMCC which creates 4-(N-maleimidomethylcyclohexane)-1-amidyl-protein (MCA-protein) covalent linkages with the primary amino groups of proteins. In the second step, cell-MCA-protein conjugates are rapidly prepared (in less than 30 min) by reacting MCA-protein with 'reduced' cells which have been pre-incubated (for about 1 h) with dithiothreitol (DTT). Stable protein-cell conjugates result from the covalent thioether linkages formed between the maleimido groups of the derivatized protein and the cell surface thiol groups created by DTT reduction. Protein-cell conjugates have been made in this way with several different proteins using several different types of cells. Such conjugates are characterized in this study by complement plus antibody-mediated cytotoxicity (CAMC) and by antibody-dependent cellular cytotoxicity (ADCC) with human effector lymphocytes. Because these protein-cell conjugates are shown to remain viable and to retain significant levels of coupled protein for at least 24 h in culture, they are potentially useful for probing various membrane-related phenomena such as the recognition of cell surface antigens by immune effector cells.
D000818:Animals; D000920:Antibody-Dependent Cell Cytotoxicity; D002460:Cell Line; D002462:Cell Membrane / Q000276:immunology / Q000378:metabolism; D003165:Complement System Proteins / Q000276:immunology; D003432:Cross-Linking Reagents; D003574:Cytochrome c Group / Q000378:metabolism*; D003602:Cytotoxicity, Immunologic; D008214:Lymphocytes / Q000276:immunology*; D008223:Lymphoma / Q000276:immunology; D008301:Maleimides; D051379:Mice; D008807:Mice, Inbred BALB C; D011485:Protein Binding
D003432:Cross-Linking Reagents; D003574:Cytochrome c Group; D008301:Maleimides; C025285:N-(4-carboxycyclohexylmethyl)maleimide N-hydroxysuccinimide ester; D003165:Complement System Proteins