Medizinische Universität Graz Austria/Österreich - Forschungsportal - Medical University of Graz
Gewählte Publikation:
Grigorenko, V; Andreeva, I; Börchers, T; Spener, F; Egorov, A.
A genetically engineered fusion protein with horseradish peroxidase as a marker enzyme for use in competitive immunoassays.
Anal Chem. 2001; 73(6):1134-1139
Doi: 10.1021/ac000684t
Web of Science
PubMed
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- Co-Autor*innen der Med Uni Graz
- Spener Friedrich
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- Abstract:
- Horseradish peroxidase is one of the most widely used marker enzymes in immunoassays. Several disadvantages are encountered upon chemical conjugation of peroxidase with antibodies or antigens, as are low reproducibility and undefined stoichiometry. We here describe for the first time the production of a recombinant fusion of a protein analyte with horseradish peroxidase in Escherichia coli, employing refolding of inclusion bodies and reconstitution with heme. The genetic fusion approach enables preparation of conjugates with 1:1 stoichiometry and defined structure. As a protein analyte, the human heart fatty acid binding protein (H-FABP) was chosen, which is a new and sensitive marker for acute myocardial infarction. The recombinant conjugate was fully active [650 U/mg with 2,2-azino-bis(3-ethyl-thiazoline-6-sulfonate) as substrate] and obtained in a yield of 12 mg/L of E. coli culture, which is better than that for recombinant peroxidase alone. The competitive immunoassay that was developed with the recombinant conjugate requires fewer incubation steps than the traditional sandwich ELISA format. It permitted the detection of H-FABP directly in plasma in the range of 10-1500 ng/mL which is the relevant range for clinical decision-making.
- Find related publications in this database (using NLM MeSH Indexing)
- Biological Markers - analysis
- Carrier Proteins - chemistry Carrier Proteins - genetics
- Cloning, Molecular -
- Escherichia coli - genetics Escherichia coli - metabolism
- Fatty Acid-Binding Proteins -
- Horseradish Peroxidase - chemistry Horseradish Peroxidase - genetics
- Humans -
- Immunoassay - methods
- Myocardial Infarction - diagnosis
- Neoplasm Proteins -
- Recombinant Fusion Proteins - chemistry Recombinant Fusion Proteins - metabolism
- Tumor Suppressor Proteins -