Dual detection of peptides in a fluorescence binding assay by employing genetically fused GFP and BFP mutants

Jennifer C. Lewis, Sylvia Daunert

Research output: Contribution to journalArticle

15 Citations (Scopus)

Abstract

A competitive fluorescence microplate assay based on a red-shifted green fluorescent protein (rsGFP) and a blue fluorescent protein (BFP) was developed for the detection of two model peptides in the same sample. The assay employed gene fusion to prepare the fluorescently labeled peptide conjugates. Specifically, plasmids were constructed in which the genes encoding for the two small peptides (less than 12 amino acids in length) were fused to either the gene of the rsGFP or the BFP, as desired. The newly constructed plasmids were transformed into E. coli for expression of the fusion proteins. By employing the technique of gene fusion, one-to-one homogeneous populations of peptide-rsGFP or -BFP conjugates were produced. These pepfide-GFP mutant conjugates exhibited the same excitation and emission spectral characteristics as the unmodified proteins. The naturally fluorescent proteins act as labels to provide sensitive dual detection of the two selected small peptides in a competitive assay format. To our knowledge, this is the first time that mutants of GFP, such as the rsGFP and BFP, have been used as quantitative labels for the development of a dual-analyte fluorescence immunoassay.

Original languageEnglish
Pages (from-to)4321-4327
Number of pages7
JournalAnalytical Chemistry
Volume71
Issue number19
DOIs
StatePublished - Oct 1 1999
Externally publishedYes

Fingerprint

Mutant Proteins
Assays
Fluorescence
Peptides
Green Fluorescent Proteins
Proteins
Fusion reactions
Genes
Labels
Plasmids
Gene encoding
Escherichia coli
Amino Acids

ASJC Scopus subject areas

  • Analytical Chemistry

Cite this

Dual detection of peptides in a fluorescence binding assay by employing genetically fused GFP and BFP mutants. / Lewis, Jennifer C.; Daunert, Sylvia.

In: Analytical Chemistry, Vol. 71, No. 19, 01.10.1999, p. 4321-4327.

Research output: Contribution to journalArticle

@article{edb9f6a6e29f4a639df80d9da529ed17,
title = "Dual detection of peptides in a fluorescence binding assay by employing genetically fused GFP and BFP mutants",
abstract = "A competitive fluorescence microplate assay based on a red-shifted green fluorescent protein (rsGFP) and a blue fluorescent protein (BFP) was developed for the detection of two model peptides in the same sample. The assay employed gene fusion to prepare the fluorescently labeled peptide conjugates. Specifically, plasmids were constructed in which the genes encoding for the two small peptides (less than 12 amino acids in length) were fused to either the gene of the rsGFP or the BFP, as desired. The newly constructed plasmids were transformed into E. coli for expression of the fusion proteins. By employing the technique of gene fusion, one-to-one homogeneous populations of peptide-rsGFP or -BFP conjugates were produced. These pepfide-GFP mutant conjugates exhibited the same excitation and emission spectral characteristics as the unmodified proteins. The naturally fluorescent proteins act as labels to provide sensitive dual detection of the two selected small peptides in a competitive assay format. To our knowledge, this is the first time that mutants of GFP, such as the rsGFP and BFP, have been used as quantitative labels for the development of a dual-analyte fluorescence immunoassay.",
author = "Lewis, {Jennifer C.} and Sylvia Daunert",
year = "1999",
month = "10",
day = "1",
doi = "10.1021/ac990404d",
language = "English",
volume = "71",
pages = "4321--4327",
journal = "Analytical Chemistry",
issn = "0003-2700",
publisher = "American Chemical Society",
number = "19",

}

TY - JOUR

T1 - Dual detection of peptides in a fluorescence binding assay by employing genetically fused GFP and BFP mutants

AU - Lewis, Jennifer C.

AU - Daunert, Sylvia

PY - 1999/10/1

Y1 - 1999/10/1

N2 - A competitive fluorescence microplate assay based on a red-shifted green fluorescent protein (rsGFP) and a blue fluorescent protein (BFP) was developed for the detection of two model peptides in the same sample. The assay employed gene fusion to prepare the fluorescently labeled peptide conjugates. Specifically, plasmids were constructed in which the genes encoding for the two small peptides (less than 12 amino acids in length) were fused to either the gene of the rsGFP or the BFP, as desired. The newly constructed plasmids were transformed into E. coli for expression of the fusion proteins. By employing the technique of gene fusion, one-to-one homogeneous populations of peptide-rsGFP or -BFP conjugates were produced. These pepfide-GFP mutant conjugates exhibited the same excitation and emission spectral characteristics as the unmodified proteins. The naturally fluorescent proteins act as labels to provide sensitive dual detection of the two selected small peptides in a competitive assay format. To our knowledge, this is the first time that mutants of GFP, such as the rsGFP and BFP, have been used as quantitative labels for the development of a dual-analyte fluorescence immunoassay.

AB - A competitive fluorescence microplate assay based on a red-shifted green fluorescent protein (rsGFP) and a blue fluorescent protein (BFP) was developed for the detection of two model peptides in the same sample. The assay employed gene fusion to prepare the fluorescently labeled peptide conjugates. Specifically, plasmids were constructed in which the genes encoding for the two small peptides (less than 12 amino acids in length) were fused to either the gene of the rsGFP or the BFP, as desired. The newly constructed plasmids were transformed into E. coli for expression of the fusion proteins. By employing the technique of gene fusion, one-to-one homogeneous populations of peptide-rsGFP or -BFP conjugates were produced. These pepfide-GFP mutant conjugates exhibited the same excitation and emission spectral characteristics as the unmodified proteins. The naturally fluorescent proteins act as labels to provide sensitive dual detection of the two selected small peptides in a competitive assay format. To our knowledge, this is the first time that mutants of GFP, such as the rsGFP and BFP, have been used as quantitative labels for the development of a dual-analyte fluorescence immunoassay.

UR - http://www.scopus.com/inward/record.url?scp=0033214807&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0033214807&partnerID=8YFLogxK

U2 - 10.1021/ac990404d

DO - 10.1021/ac990404d

M3 - Article

C2 - 10517149

AN - SCOPUS:0033214807

VL - 71

SP - 4321

EP - 4327

JO - Analytical Chemistry

JF - Analytical Chemistry

SN - 0003-2700

IS - 19

ER -