title: Endogenous biotin-binding proteins: an overlooked factor causing false positives in streptavidin-based protein detection.
abstract: Biotinylation is widely used in DNA, RNA and protein probing assays as this molecule has generally no impact on the biological activity of its substrate. During the streptavidin-based detection of glycoproteins in Lactobacillus rhamnosus GG with biotinylated lectin probes, a strong positive band of approximately 125 kDa was observed, present in different cellular fractions. This potential glycoprotein reacted heavily with concanavalin A (ConA), a lectin that specifically binds glucose and mannose residues. Surprisingly, this protein of 125 kDa could not be purified using a ConA affinity column. Edman degradation of the protein, isolated via cation and anion exchange chromatography, lead to the identification of the band as pyruvate carboxylase, an enzyme of 125 kDa that binds biotin as a cofactor. Detection using only the streptavidin conjugate resulted in more false positive signals of proteins, also in extracellular fractions, indicating biotin-associated proteins. Indeed, biotin is a known cofactor of numerous carboxylases. The potential occurence of false positive bands with biotinylated protein probes should thus be considered when using streptavidin-based detection, e.g. by developing a blot using only the streptavidin conjugate. To circumvent these false positives, alternative approaches like detection based on digoxigenin labelling can also be used.
authors.affiliation: Department 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.
authors.forename: Hanne L P
authors.initials: HL
authors.lastname: Tytgat
authors.forename: Geert
authors.initials: G
authors.lastname: Schoofs
authors.forename: Michèle
authors.initials: M
authors.lastname: Driesen
authors.forename: Paul
authors.initials: P
authors.lastname: Proost
authors.forename: Els J M
authors.initials: EJ
authors.lastname: Van Damme
authors.forename: Jos
authors.initials: J
authors.lastname: Vanderleyden
authors.forename: Sarah
authors.initials: S
authors.lastname: Lebeer
mesh_terms: D001426:Bacterial Proteins / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D001710:Biotin / Q000032:analysis / Q000378:metabolism; D019921:Biotinylation; D015153:Blotting, Western / Q000379:methods / Q000592:standards*; D002352:Carrier Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D052201:Lacticaseibacillus rhamnosus / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D019809:Streptavidin / Q000032:analysis* / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D002352:Carrier Proteins; C017071:biotin-binding proteins; D001710:Biotin; D019809:Streptavidin