Purification, physicochemical and immunological characterization of arginine kinase, an allergen of crayfish (Procambarus clarkii).
Arginine kinase (AK) has attracted considerable attention because it has been identified as a shellfish allergen. However, little information is available about AK in crayfish (Procambarus clarkii). In this study, crayfish AK was purified and cloned. Its physicochemical properties, processing stability, and immunological characteristics were analyzed. Crayfish AK was purified by column chromatography, which revealed a single band with molecular mass of 40 kDa; this result was further confirmed by mass spectrometry. The full-length gene sequence of crayfish AK was 1462 bp and encoded a protein of 357 amino acid residues. The results of this study revealed that crayfish AK is a glycoprotein with an isoelectric point of approximately 6.5. Thermal stability assays revealed that crayfish AK easily forms aggregates at temperatures >44°C and was stable at pH 4.0-8.0. SDS-PAGE and dot blotting were used to assess processing stability of purified AK. The results revealed that the IgE-binding activity of crayfish AK is reduced after boiling.
College of Biological Engineering, The Key Laboratory of Science and Technology for Aquaculture and Food Safety, Jimei University, 43 Yindou Road, Xiamen 361021, Fujian, China.
Heng-Li
HL
Chen
Hai-Yan
HY
Mao
Min-Jie
MJ
Cao
Qiu-Feng
QF
Cai
Wen-Jin
WJ
Su
Yong-Xia
YX
Zhang
Guang-Ming
GM
Liu
D000485:Allergens / Q000276:immunology* / Q000302:isolation & purification; D000595:Amino Acid Sequence; D000818:Animals; D001122:Arginine Kinase / Q000737:chemistry* / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification* / Q000378:metabolism; D003400:Astacoidea / Q000276:immunology; D001483:Base Sequence; D003001:Cloning, Molecular; D004795:Enzyme Stability; D006801:Humans; D007106:Immune Sera / Q000378:metabolism; D007073:Immunoglobulin E / Q000378:metabolism; D007526:Isoelectric Point; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011487:Protein Conformation; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D000485:Allergens; D007106:Immune Sera; D007073:Immunoglobulin E; D001122:Arginine Kinase
AK; Allergen; Arginine kinase; ELISA; Immunological characterization; Physicochemical properties; Procambarus clarkii; Purification; SDS–PAGE; SDS–polyacrylamide gel electrophoresis; SGF; SIF; arginine kinase; enzyme-linked immunosorbent assay; isoelectric point; pI; simulated gastric fluid; simulated intestinal fluid