Purification, Physicochemical Properties, and Mimotope Peptide Prediction of Tropomyosin from Antarctic Krill (Euphausia superba)

Tropomyosin (TM) from Antarctic krill (Euphausia superba) was extracted, purified, and identified by mass spectrometry, and its thermal stability, pH stability and digestive stability were studied. Meanwhile, sequence homology analysis, spatial structure homology modeling, and allergen mimotope peptide recognition were carried out using bioinformatics. The crude protein was extracted, thermally treated to remove impurities, precipitated at the isoelectric point and purified by salting-out. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) of the purified protein showed only one clear band with molecular mass near 32 kDa. The target protein was identified as TM (Euphausia superba) by ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) analysis and UniProt database search. The molecular mass of the protein was 32.6 kDa and the peptide coverage was 97%. The results of homology analysis and physicochemical properties showed that TM from Antarctic krill was a highly conserved protein, and its sequence homology with those from 26 crustacean species ranged from 88% to 98.2%. TM from Antarctic krill had strong stability to heat, acid, alkali and gastric juice digestion, but poor stability to intestinal juice digestion, and was easily degraded by trypsin and chymotrypsin to produce low-molecular-mass peptides. Eight mimotope peptides (EAQNKETNAKADKADDEVH, DLERSEERLN, TKLAEASQAADESER, EADRKYDE, ERAEERAEAG, VSEEKANQREEAYKEQI, RSVQKLQKEVDR, VNEKEKYKGI) in Antarctic krill TM were finally predicted and identified by five bioinformatics tools including DNAStar Protean, ANTHEPROT, BepiPred 1.0 server, ABCpred server, and Immunomedicine Group, and mapped in the spatial structure. This study provides a scientific basis for the accurate prediction and identification of mimotope peptides in TM from Antarctic krill and the development of hypoallergenic products.