title: Solid phase assembly of defined protein conjugates.
abstract: We have developed a solid-phase procedure for protein-protein conjugation that gives greater control over product size and composition than previous methods. Conjugates are assembled by sequential addition of activated proteins to the support under conditions suitable for maintaining the activity of the proteins. The total number of conjugate units to be prepared is fixed in the first step by the quantity of the first protein absorbed by the support. In each following step, the added protein links only to previously bound protein. The final conjugate is released to solution by cleaving the linker holding the first protein to the support. This stepwise assembly provides uniformly sized conjugates of the desired size and composition with placement of components at the desired positions within the structure. Using this approach, we have prepared a series of conjugates containing R-phycoerythrin as the central protein, with varying quantities of alkaline phosphatase and IgG with expected molecular masses ranging from 1.6 to 11.5 MDa. Size-exclusion chromatography and atomic force microscopy demonstrate homogeneity and control of the conjugate size. In an immunoassay for human thyroid stimulating hormone, the conjugates show signals consistent with their compositions.
authors.affiliation: D09FQ, AP20, Abbott Laboratories, 100 Abbott Park Road, Abbott Park, Illinois 60064-6015, USA. john.c.russell@abbott.com
authors.forename: John C
authors.initials: JC
authors.lastname: Russell
authors.forename: Tracey L
authors.initials: TL
authors.lastname: Colpitts
authors.forename: Shelley R
authors.initials: SR
authors.lastname: Holets-McCormack
authors.forename: Thomas G
authors.initials: TG
authors.lastname: Spring
authors.forename: Stephen D
authors.initials: SD
authors.lastname: Stroupe
authors.forename: Andrew D
authors.initials: AD
authors.lastname: Vogt
authors.forename: Sie-Ting
authors.initials: ST
authors.lastname: Wong
mesh_terms: D000469:Alkaline Phosphatase / Q000737:chemistry; D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry; D002851:Chromatography, High Pressure Liquid; D020650:Combinatorial Chemistry Techniques; D003432:Cross-Linking Reagents / Q000737:chemistry; D019281:Dimerization; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D018796:Immunoconjugates / Q000737:chemistry*; D007074:Immunoglobulin G / Q000737:chemistry; D008301:Maleimides / Q000737:chemistry; D018625:Microscopy, Atomic Force; D010799:Phycoerythrin / Q000737:chemistry; D011506:Proteins / Q000737:chemistry*; D013972:Thyrotropin / Q000032:analysis
chemical_list: D000911:Antibodies, Monoclonal; D003432:Cross-Linking Reagents; D018796:Immunoconjugates; D007074:Immunoglobulin G; D008301:Maleimides; D011506:Proteins; D010799:Phycoerythrin; D013972:Thyrotropin; D000469:Alkaline Phosphatase