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Bacterial Peptide Display for the Selection of Novel Biotinylating Enzymes
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Endogenous biotin-binding proteins: an overlooked factor causing false positives in streptavidin-based protein
Hanne L P Tytgat1, Geert Schoofs, Michèle Driesen
1Department of Bioscience Engineering, Research Group Environmental Ecology and Applied Microbiology, University of Antwerp, Antwerp, Belgium; Department of Microbial and Molecular Systems, Centre of Microbial and Plant Genetics, Leuven, Belgium.
Microbial Biotechnology
|September 12, 2014
Summary
Biotinylated probes can cause false positives in streptavidin-based detection. Pyruvate carboxylase, a biotin-dependent enzyme, was mistakenly identified as a glycoprotein in Lactobacillus rhamnosus GG.
Area of Science:
- Microbiology
- Biochemistry
- Molecular Biology
Background:
- Biotinylation is a common technique for labeling biomolecules in assays.
- Streptavidin-biotin interaction is widely used for detection.
- Glycoprotein detection in Lactobacillus rhamnosus GG was investigated.
Purpose of the Study:
- To investigate a strong positive band observed during streptavidin-based detection of glycoproteins.
- To identify the protein responsible for the observed signal.
- To assess the reliability of biotinylated probes in streptavidin-based detection.
Main Methods:
- Streptavidin-based detection with biotinylated lectin probes.
- Concanavalin A (ConA) affinity chromatography.
- Cation and anion exchange chromatography.
- Edman degradation for protein identification.
Main Results:
- A 125 kDa band, initially suspected to be a glycoprotein, was detected.
- Edman degradation identified the band as pyruvate carboxylase, a biotin-dependent enzyme.
- Streptavidin-only detection revealed numerous false positive signals, including extracellular proteins.
Conclusions:
- Pyruvate carboxylase can be misidentified as a glycoprotein in streptavidin-based detection assays.
- Biotin-associated proteins can lead to false positive signals.
- Alternative detection methods like digoxigenin labeling or using only streptavidin conjugate are recommended to avoid false positives.
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