Genetically Engineered Obelin as a Bioluminescent Label in an Assay for a Peptide

Матвеев, С. В.; Lewis, John L.; Daunert, Sylvia

doi:10.1006/abio.1999.4056

Cited by 6 publications

(1 citation statement)

References 22 publications

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“…Recently, we prepared one-to-one photoprotein-labeled analytes by fusing an octapeptide to the N-terminus of the photoproteins, aequorin and obelin. These highly reproducible homogeneous conjugates yielded excellent detection limits for the peptide even in a system with a relatively low binding constant between the antigen and the antibody ( K D ≅ 10 -6 ). , In this study, we have expanded the range of possible analytes for the production of desired homogeneous conjugates with aequorin to those that are nonpeptidic in nature, e.g., hormones, drugs, and vitamins, by genetically modifying the photoprotein. Thyroxine, a hormone used to monitor patient thyroid status, was selected as a model analyte for the site-specific conjugation to the aequorin mutants containing unique cysteine residues.…”

Section: Resultsmentioning

confidence: 99%

Bioluminescence Immunoassay for Thyroxine Employing Genetically Engineered Mutant Aequorins Containing Unique Cysteine Residues

Lewis

Daunert

2001

Anal. Chem.

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Genetically engineered one-to-one conjugates between an analyte and a protein label have been demonstrated to yield assays with better detection limits and performance characteristics than those prepared by conventional chemical conjugation methods. To date, the preparation of these conjugates has been limited to fusion techniques where a peptide analyte is fused in frame to the protein label. To further expand the range of analytes that can be detected by using genetic engineering techniques coupled with bioanalytical methods, we have employed site-directed mutagenesis to prepare one-to-one analyte-label conjugates that include nonpeptidic analytes such as drugs, vitamins, and hormones. Specifically, we have prepared mutants of the photoprotein aequorin containing single cysteine residues suitable for site-specific conjugation. Aequorin is a photoprotein that emits light at 469 nm and has been employed as a highly sensitive bioluminescent label in the development of binding assays for important biomolecules. We have performed polymerase chain reaction-based site-directed mutagenesis on apoaequorin to yield four mutant aequorins containing unique cysteine residues at positions 5, 53, 71, and 84 in the polypeptide chain for the purpose of site-specific conjugation to a model analyte. A maleimide-activated thyroxine was selected as the model analyte and site-specifically conjugated to the mutants through their unique cysteine residues. A heterogeneous assay for thyroxine was then developed by employing the genetically engineered aequorin mutants.

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Section: Resultsmentioning

confidence: 99%

Bioluminescence Immunoassay for Thyroxine Employing Genetically Engineered Mutant Aequorins Containing Unique Cysteine Residues

Lewis

Daunert

2001

Anal. Chem.

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Luminescent Proteins in Binding Assays

Roda

Guardigli

Michelini

et al. 2008

Protein Science Encyclopedia

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Originally published in: Photoproteins in Bioanalysis. Edited by Sylvia Daunert and Sapna K. Deo. Copyright © 2006 Wiley‐VCH Verlag GmbH & Co. KGaA Weinheim. Print ISBN: 3‐527‐31016‐6 The sections in this article are Introduction Protein–Protein and Protein–Ligand Interaction Assays FRET and BRETM Techniques FRET and BRET Applications Other Detection Principles Antibody‐based Binding Assays Chemical Conjugation Gene Fusion Dual‐analyte Assays Expression Immunoassays BRET‐based Immunoassays Biotin–Avidin Binding Assays Nucleic Acid Hybridization Assays Other Binding Assays Conclusions

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