Antigenicity of recombinant proteins after regioselective immobilization onto polyanhydride-based copolymers.
We previously demonstrated that the introduction of a tag consisting of several contiguous lysines at the N- or C-terminus of a recombinant protein greatly improved the covalent grafting of the protein onto negatively charged maleic anhydride-alt-methyl vinyl ether (MAMVE) copolymer, under many different experimental conditions (Ladavière, C., et al. (1998) Bioconjugate Chem. 9, 655; Allard, L., et al. (2002) Biotechnol. Bioeng. 80, 341). The grafting efficiency was dependent on the charge and amine density of the tag, characteristics which were determined by the tag composition. The six lysine tag (Lys6) was found to be the most efficient (Allard, L., et al. (2001) Bioconjugate Chem. 12, 972). In the present work, the biological activity of Lys6-proteins covalently bound to polymer was investigated. N- or C-terminal Lys6-tagged HIV-1 p24 recombinant proteins (RK24H and RH24K) were grafted onto MAMVE, and the antigenicity each of the bioconjugates was evaluated using six monoclonal antibodies that recognized different epitopes distributed along the protein. We demonstrate that the position of the tag and the hydrolysis rate of the anhydride moieties of the polymer are the two main parameters involved in the conservation of the biological activity of the immobilized protein. We thus present a process which allows an efficient oriented immobilization of proteins onto copolymers with optimal biological activity that is suitable for the controlled production of active bioconjugates.
Allard
Laure
L
Unité Mixte de Recherche 2714, CNRS-bioMérieux, IFR128 Biosciences Lyon-Gerland, ENS-Lyon, 46, allée d'Italie, 69364 Lyon, France.
Cheynet
Valérie
V
Oriol
Guy
G
Gervasi
Gaspard
G
Imbert-Laurenceau
Emmanuelle
E
Mandrand
Bernard
B
Delair
Thierry
T
Mallet
François
F
D000595:Amino Acid Sequence; D000812:Anhydrides / Q000737:chemistry*; D000939:Epitopes; D016655:HIV Core Protein p24 / Q000737:chemistry / Q000276:immunology; D055767:Immobilized Proteins; D008239:Lysine / Q000737:chemistry; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D011108:Polymers / Q000737:chemistry*; D011994:Recombinant Proteins / Q000737:chemistry* / Q000276:immunology* / Q000302:isolation & purification; D013237:Stereoisomerism
D000812:Anhydrides; D000939:Epitopes; D016655:HIV Core Protein p24; D055767:Immobilized Proteins; D011108:Polymers; D011994:Recombinant Proteins; D008239:Lysine