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.