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

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.

Keywords

• jug r 1; walnut allergenic protein; separation and purification; identification; structural characterization