Expression and purification of 15N-labeled Fra a 1, a strawberry allergen, to prepare samples for NMR measurements.
Protein Expr Purif
; 210: 106296, 2023 10.
Article
em En
| MEDLINE
| ID: mdl-37192728
ABSTRACT
Raw strawberries contain allergens that cause oral allergic syndrome. Fra a 1 is one of the major allergens in strawberries and might decrease their allergenicity by heating, likely due to structural changes in the allergen leading to decreased recognition of the allergens in the oral cavity. In the present study, to understand the relationship between allergen structure and allergenicity, the expression and purification of 15N-labeled Fra a 1 were examined and the sample was used for NMR analysis. Two isoforms, Fra a 1.01 and Fra a 1.02, were used and expressed in E. coli BL21(DE3) in M9 minimal medium. Fra a 1.02 was purified as a single protein by using the GST tag approach, whereas histidine × 6-tag (his6-tag) Fra a 1.02 was obtained both as the full-length (â¼20 kDa) and a truncated (â¼18 kDa) form. On the other hand, his6-tag Fra a 1.01 was purified as a homogeneous protein. 15N-labeled HSQC NMR spectra suggested that Fra a 1.02 was thermally denatured at lower temperatures than Fra a 1.01, despite the high amino acid sequence homology (79.4%) of these isoforms. Furthermore, the samples in the present study allowed us to analyze ligand binding that probably affects structural stability. In conclusion, GST tag was effective for obtaining a homogeneous protein when his6-tag failed to give a single form, and the present study provided a sample that could be used for NMR studies of the details of the allergenicity and structure of Fra a 1.
Palavras-chave
Texto completo:
1
Base de dados:
MEDLINE
Assunto principal:
Alérgenos
/
Fragaria
Idioma:
En
Revista:
Protein Expr Purif
Assunto da revista:
BIOLOGIA MOLECULAR
Ano de publicação:
2023
Tipo de documento:
Article
País de afiliação:
Japão