FOOD SCIENCE ›› 2023, Vol. 44 ›› Issue (20): 127-135.doi: 10.7506/spkx1002-6630-20230408-070

• Bioengineering • Previous Articles     Next Articles

Isolation, Purification, Identification, and Analysis of Jug r 1, a Major Allergenic Protein in Walnut

SHEN Mingjuan, LI Yunqian, YANG Xi, WANG Yongqin, SHA Xiaomei, ZHANG Xuechun   

  1. (1. College of Life Science, Southwest Forestry University, Kunming 650224, China; 2. National R&D Center for Freshwater Fish Processing, College of Life Sciences, Jiangxi Normal University, Nanchang 330022, China; 3. Key Laboratory of Forest Disaster Warning and Control of Yunnan Province, Southwest Forestry University, Kunming 650224, China)
  • Online:2023-10-25 Published:2023-11-07

Abstract: In this study, by using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and double-sandwich enzyme-linked immunosorbent assay (ELISA), Juglans sigillata kernels from seven different production areas in Yunnan province were screened for their Jug r 1 contents. The preparation of Jug r 1 through ammonium sulfate precipitation, gel filtration chromatography (GFC), and liquid chromatography-tandem mass spectrometry (LC-MS/MS) was optimized and the product was identified. Its structure was characterized by circular dichroism (CD) spectroscopy. Finally, Jug r 1 content in walnut protein was measured by ELISA. The results showed that walnuts from Baoshan, which had the highest contents of total protein and Jug r 1, were used for subsequent experiments. The optimal concentration of ammonium sulfate saturation was in the range from 40% to 80%, and the optimal conditions for GFC were sample concentration of 30 mg/mL, loading volume of 4 mL, and elution flow rate of 1 mL/min. Under these conditions, the recovery of Jug r 1 was 16.58%. Mass spectrometry analysis showed that this protein had the typical characterisitcs of Jug r 1. Circular dichroism spectroscopy indicated that the secondary structure of Jug r 1 was dominated by α-helix and consisted of multiple conformations. After separation and purification by a two-step process, Jug r 1 with purity more than 96% was obtained. This study can provide a scientific basis for further research on Jug r 1 and also provide a reference for the separation and purification of allergic proteins from other nuts.

Key words: Jug r 1; walnut allergenic protein; separation and purification; identification; structural characterization

CLC Number: