A Genetically Engineered Fusion Protein with Horseradish Peroxidase as a Marker Enzyme for Use in Competitive Immunoassays

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• 作者：Vitaly Grigorenko ; Irina Andreeva ; Torsten Bö ; rchers ; Friedrich Spener ; and Alexey Egorov
• 刊名：Analytical Chemistry
• 出版年：2001
• 出版时间：March 15, 2001
• 年：2001
• 卷：73
• 期：6
• 页码：1134 - 1139
• 全文大小：101K
• 年卷期：v.73,no.6(March 15, 2001)
• ISSN：1520-6882

文摘

Horseradish peroxidase is one of the most widely usedmarker enzymes in immunoassays. Several disadvantagesare encountered upon chemical conjugation of peroxidasewith antibodies or antigens, as are low reproducibility andundefined stoichiometry. We here describe for the firsttime the production of a recombinant fusion of a proteinanalyte with horseradish peroxidase in Escherichia coli,employing refolding of inclusion bodies and reconstitutionwith heme. The genetic fusion approach enables preparation of conjugates with 1:1 stoichiometry and definedstructure. As a protein analyte, the human heart fatty acidbinding protein (H-FABP) was chosen, which is a new andsensitive marker for acute myocardial infarction. Therecombinant conjugate was fully active [650 U/mg with2,2-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 peroxidasealone. The competitive immunoassay that was developedwith the recombinant conjugate requires fewer incubationsteps than the traditional sandwich ELISA format. Itpermitted the detection of H-FABP directly in plasma inthe range of 10-1500 ng/mL which is the relevant rangefor clinical decision-making.