Versatile method for production and controlled polymer-immobilization of biologically active recombinant proteins.
The immobilization of a protein by covalent attachment to a support matrix should involve only functional groups of the protein that are not essential for its biological activity. A general strategy for obtaining recombinant proteins designed for oriented covalent grafting onto copolymers was investigated. The rationale involves the definition of seven p24-derived recombinant proteins as fused to either distant or adjacent tags comprising primary amine rich tag consisting of six contiguous lysines suitable for oriented covalent immobilization and a hexa-histidine tag suitable for metal chelate affinity purification. High-level expression, efficient affinity purification, and coupling yields onto maleic anhydride-alt-methyl vinyl ether copolymers higher than 95% were obtained for all proteins. Afterwards, an investigation of the biological features of the immobilized vs. nonimmobilized protein onto the copolymer allowed us to select one bioconjugate which was used in a diagnostic context, i.e., as a capture antigen in an ELISA format test. Sera from 107 HIV-seropositive individuals at various stages of HIV infection, including two seroconversion panels and 104 healthy HIV-seronegative controls, were tested using either RH24 or RK24H-copolymer coated onto the microtiter plate. These assays showed that the use of such a protein-copolymer bioconjugate allowed detection of lower antibody titers than the RH24 protein, illustrating the potential of applications of such doubly tagged proteins. Thus, a set of expression vectors was designed containing four different combinations of hexa-lysine and hexa-histidine tags and a multiple cloning site, allowing the production of different recombinant fusion proteins suitable for biological reactivity conservation after immobilization.
Allard
Laure
L
Unité Mixte UMR 2142 CNRS-bioMérieux, ENS-Lyon, 46, allée d'Italie, 69364 Lyon, France.
Cheynet
Valérie
V
Oriol
Guy
G
Mandrand
Bernard
B
Delair
Thierry
T
Mallet
François
F
D000595:Amino Acid Sequence; D001483:Base Sequence; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D015870:Gene Expression; D016655:HIV Core Protein p24 / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification; D006801:Humans; D008957:Models, Genetic; D008969:Molecular Sequence Data; D011108:Polymers; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D012680:Sensitivity and Specificity; D016324:Sequence Tagged Sites
D016655:HIV Core Protein p24; D011108:Polymers; D011994:Recombinant Proteins