Objective
Infection with the AlDS-causing human immunodeficiency virus (HIV) still presents one of the current major challenges to mankind. In the context of the wide-spread occurrence of HIV infection, the long asymptomatic carrier state and the lethality of the disease, it is highly desirable to have access to fast, reliable and inexpensive assays to detect anti-HlV antibodies in HlV-infected individuals. The aim of this proposal is to develop new diagnostic probes for simple and fast detection of anti-HlV antibodies in human plasma. The proposed work is based on the previous discovery of Partner 2 that the insertion of antigenic peptides in specihc sites of the bacterial enzyme beta-galactosidase causes a severe reduction of the enzymatic activity, which can be partially recovered upon antibody binding to the inserted antigenic peptide (Benito et al., 1996, J. Biol. Chem. 271, 21251). This phenomenon will be exploited to generate new beta-galactosidase HIV antigenic fusion proteins as enzymatic probes for anti-HlV antibody detection. The recovery of beta-galactosidase activity upon antigen-antibody binding will allow the detection of antibodies by simple colorimetric quantification of beta-galactosidase activity in a homogeneous test system, involving only the antigenic fusion protein, the chromogenic substrate and a serum sample for testing. The beta-galactosidase enzyme (EC 3.2.1.23) encoded by the Eschenchia coli lacZ gene, is a high molecular mass multimeric protein composed of four non-covalently linked subunits. Analysis of beta-galactosidase activity is accomplished by simple colorimetric assays (Miller, 1972, Experiments in Molecular Genetics, Cold Spring Harbour Laboratory, p. 352) and small-scale purification of the enzyme can be carried out in one-step purification procedures (Ullmann, 1984, Gene 29, 27). beta-Galactosidase and recombinant beta-galactosidase fusion proteins are in general stable, non-toxic for the producing cells, and they can be produced in high yields in recombinant bacteria. Moreover, beta-galactosidase does not react with human serum rendering this enzyme ideal for the design of new molecular probes for diagnosis of human infectious diseases. Partner 4, an important European industrial company with a wide and recognized expertise in immunodiagnostics, has suggested specific HIV Env antigenic peptides as a model of commercial relevance for the design, construction and improvement of beta-galactosidase molecular probes. Partner 2, a research laboratory devoted to the design and production of recombinant proteins, will construct fusion proteins with different HIV Env peptides inserted within the activating loop of the enzyme and determine their enzymatic properties. Among those constructs, protein chimeras responsive to antibody-mediated reactivation will be transferred to Partner 3. This research laboratory, mainly working on high cell density production of recombinant proteins and protein characterization, will establish medium- and large-scale production and purification protocols for these proteins, characterize the antibody binding parameters in different constructs and also determine the effects of the peptide insertion on the in vivo and in vitro stability of the chimeric proteins. The most interesting proteins will be transferred to Partner 4 for the evaluation of the diagnostic feasibility of the concept. Partner 2 will also transfer the amino acid sequences of both the responsive and non-responsive proteins to Partner 3. This research group is devoted to the modeling of protein structure. Conformational studies will be done by this partner on selected constructs, focusing on the activating interface and the insertion sites. Three-dimensional data of native beta-galactosidase obtained in Prof. Matthews' laboratory (Jacobson et al. 1994, Nature 369, 761) will be used for this purpose. The aim of this task is to explore the structural basis of reactivation upon antibody binding, to provide a rational for the construction of new modified mutants, especially concerning the appropriate insert length and the insertion sites of the antigenic peptides. From an integrated exchange of information between Partners 1, 2, 3, and 4, sequence modifications on the previous constructs will be suggested to Partner 2, in order to improve critical properties of these proteins, such as increasing structural stability, reducing background activity and improving the enzymatic response. This iterative interaction will result in new sets of proteins to be tested again by Partner 4 who will evaluate these new molecular probes for their applicability in commercial homogeneous assay kits. All the partners in this collaborative research project have agreed to consider this project with high priority within their research activities.
Keywords: HIV infection, diagnostic tests, beta-galacosidase fusion proteins, recombinant Escherichia coli
Fields of science (EuroSciVoc)
CORDIS classifies projects with EuroSciVoc, a multilingual taxonomy of fields of science, through a semi-automatic process based on NLP techniques. See: The European Science Vocabulary.
CORDIS classifies projects with EuroSciVoc, a multilingual taxonomy of fields of science, through a semi-automatic process based on NLP techniques. See: The European Science Vocabulary.
- natural sciences biological sciences molecular biology molecular genetics
- natural sciences biological sciences microbiology virology
- medical and health sciences health sciences infectious diseases RNA viruses HIV
- natural sciences chemical sciences organic chemistry amines
- natural sciences biological sciences biochemistry biomolecules proteins enzymes
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Coordinator
38124 BRAUNSCHWEIG
Germany
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