Preparation of recombinant fish allergens
The protein sequences of enolase (Sal s 2; Uniprot ID: B5DGQ7) and aldolase (Sal s 3; Uniprot ID: B5DGM7) of Salmo salar weredownloaded from the Uniprot database and reverse translated by MEGA 11.0. Enolase (Accession no: GBKA01005072.1) and aldolase (Accession no: GEUQ01036650.1)
sequences of Ctenopharyngodon idella were retrieved from the transcriptome shotgun assembly deposited at GenBank (Accession no: PRJNA325430) with tBLASTn search using Sal s 2 and Sal s 3 as the search templates. The nucleotide sequences encoding the full-length allergens were commercially synthesized and cloned into the His-tag expression vector pET30(a)+. His-tagged recombinant allergens were then expressed in Escherichia coli [BL21 (DE3)] by culturing in MagicMedia (Invitrogen). Allergens were then purified using the HisPur cobalt spin columns (Thermo Scientific) as per the manufacturer’s instructions. For parvalbumin purification, raw extracts extracted in Tris-HCl (pH 8.0) were heated at 95oC – 100oC for 15 mins, followed by centrifugation to remove precipitated heat labile proteins. Supernatant containing soluble proteins were loaded into a MonoQ anion exchange column (Cytiva) connected to a NGC 10 FPLC system (Bio-Rad). Fractions containing pure parvalbumin were separated by a gradient elution of 0 – 1M NaCl in Tris-HCl (pH 8.0) and buffer exchanged into PBS for downstream applications with an Amicon centrifugal filter unit. Acid-soluble collagen was extracted from the muscle of salmon and grass carp as described previously22. Briefly, diced fish meat was resuspended in 10 volumes of 0.1mol NaOH overnight at 4°C with gentle agitation to remove non-collagenous proteins. Upon centrifugation at 3000 rpm for 10 mins and pH neutralization with ultrapure water, fish meat was soaked in 10% butanol and incubated for 24 h at 4°C with gentle agitation. After centrifugation and washing in ultrapure water, samples were resuspended in 10 volumes of 0.5M acetic acid for 4 days at 4°C with gentle agitation. The supernatant were then salted-out by adding NaCl to a final concentration of 2M. The resultant precipitate was dissolved in 0.5M acetic acid and dialyzed against distilled water.
The concentration and purity of purified fish allergens were determined on the NanoDrop OneC spectrophotometer (Thermo Scientific) and SDS-PAGE, respectively. Protein identity of the allergens was also confirmed by mass spectrometry. All purified allergens were stored at -20°C until use.