Extraction, Isolation, and Characterization of Globulin Proteins from Lupinus albus

Abstract

Lupin has recently been added to the list of allergens requiring mandatory advisory labeling on foodstuffs sold in the European Union, and since December 2008, all products containing even trace amounts of lupin must be labeled correctly. Lupin globulins consist of two major globulins called α-conglutin (11S and "legumin-like") and β-conglutin (7S and "vicilin-like") and another additional two globulins, γ-conglutin and δ-conglutin, which are present in lower amounts. We report on a methodology to facilitate the extraction of each of these proteins using centrifugation and isolation by anion-exchange chromatography followed by size-exclusion chromatography. The isolated subunits were characterized using reducing and non-reducing polyacrylamide gel electrophoresis, western blotting, and peptide mass fingerprinting, all of which revealed that the individual protein subunits are highly pure and can be used as immunogens for the production of antibodies specific for each of the conglutin fractions, as well as standards, and the extraction protocol can be used for the selective extraction of each of the subunits from foodstuffs, thus facilitating a highly accurate determination of the lupin concentration. Furthermore, the subunits can be used to elucidate information regarding the toxicity of each of the subunits, by looking at their interaction with the IgE antibodies found in the serum of individuals allergic to lupin, providing critical information for the definition of the requirements of analytical assays for the detection of lupin in foodstuffs.

DNA Aptamers against the Lup an 1 Food Allergen

“…The attachment of the ß-conglutin to magnetic beads was confirmed using peptide mass fingerprinting (PMF) using the positive controls Prostate Specific Antigen (PSA) and γ-conglutin (purified as described in [42]) with Q8IXI4 and Q9FSH9 accession number in UniProtKB and TreEMBL, respectively (Table 1). Magnetic beads blocked with ethanolamine were used as a negative control.…”

“…Here the protein attached to the magnetic bead surface was directly digested with trypsin and the peptides produced were then analyzed using peptide mass fingerprinting, and the profile obtained in each spectra was then compared to the NCBI/UniProtKB/TrEMBL databases that contain the theoretical masses derived from the in silico tryptic digestion for millions of protein sequences. According to the number of peptide masses matched, including a minimum mass error tolerance of 50 ppm and using the MOWSE score algorithm, the peptide profile in the database were ranked, and the best score identified the target protein, confirming the coupling of the magnetic beads with the ß-conglutin protein [42].…”

“…Proteins from Lupinus albus seeds were extracted, purified and characterized as previously described [42], obtaining a pure isolate of β-conglutin. β-conglutin was conjugated to Dynabeads® M-270 Carboxylic Acid magnetic beads using carbodiimide coupling.…”

Effects of autoclaving and high pressure on allergenicity of hazelnut proteins

“…The membranes were stained with red-ponceau to verify protein transfer. Western immunoblotting using pooled human sera was done, as described later, with the necessary modifications to optimize the hazelnut sample analysis for this study [33]. Blots were blocked in phosphate-buffered saline plus 0.1% Tween 20 containing 1% fat-free milk powder for 2 h at room temperature and then incubated overnight at room temperature with individual and pooled sera (1:5 dilution).…”

Purification and biochemical characterization of 11S globulin from chan (Hyptis suaveolens L. Poit) seeds

“…46, 5.56, 5.69, 5.78, 5.89 and 6.15, and those from peanut legumin have 5.41, 5.48 and 5.5 (Kottapalli et al, 2008;Natarajan et al, 2007). The helianthinin (sunflower legumin) and a-conglutin monomers have pIs of 5.0-6.0 and 5.1-5.9, respectively (Duranti et al, 2008;González-Pérez & Vereijken, 2007;Nadal, Canela, Katakis, & O'Sullivan, 2011).…”