1. Artificial intelligence-driven design of the assembled major cat allergen Fel d 1 to improve its spatial folding and IgE-reactivity.
- Author
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Zheng, Wei, Xu, Yi-Fei, Hu, Zhi-Ming, Li, Ke, Xu, Zhi-Qiang, Sun, Jin-Lyu, and Wei, Ji-Fu
- Subjects
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IMMUNOGLOBULIN E , *ALLERGENS , *ALLERGIES , *CELLULAR inclusions , *QUALITY factor , *CATS - Abstract
• We firstly provided an AI-design strategy to optimize the Fel d 1 with improved spatial conformation between two chains. • The optimized Fel d 1 could fold into its native-like structure with no extra process and its IgE-binding activity was improved. • The strategy lays the foundation for the Fel d 1's industrial production and facilitates its clinical applications for diagnosis. Cat-derived allergens are considered as one of the most common causes of allergic diseases worldwide. Fel d 1 is a major cat allergen and plays an important role in immunoglobulin E (IgE)-reaction diagnosis. However, the two separate chains of Fel d 1 exhibited lower IgE-reactivity than its complete molecule of an assembled form, which makes it difficult to efficiently prepare and limits the application of Fel d 1 in molecular diagnosis of cat allergy. We first applied artificial intelligence (AI) based tool AlphaFold2 to build the 3-dimensional structures of Fel d 1 with different connection modes between two chains, which were evaluated by ERRAT program and were expressed in Escherichia coli. We then calculated the expression ratios of soluble form/inclusion bodies form of optimized Fel d 1. The Circular Dichroism (CD), High Performance Liquid Chromatography-Size Exclusion Chromatography (HPLC-SEC) and reducing/non-reducing SDS-PAGE were performed to characterize the folding status and dimerization of the optimized fusion Fel d 1. The improvement of specific-IgE reactivity to optimized fusion Fel d 1 was investigated by enzyme linked immunosorbent assay (ELISA). Among several linkers, 2 × GGGGS got the highest scores, with an overall quality factor of 100. The error value of the residues around the junction of 2 × GGGGS was lower than others. It exhibited highest proportion of soluble protein than other Fel d 1 constructs with ERRAT (GGGGS, KK as well as direct fusion Fel d 1). The results of CD and HPLC-SEC showed the consistent folding and dimerization of two fused subunits between the optimized fusion Fel d 1 and previously well-defined direct fusion Fel d 1. The overall IgE-binding absorbance of optimized fusion Fel d 1 tested by ELISA was improved compared with that of the direct fusion Fel d 1. We firstly provided an AI-design strategy to optimize the Fel d 1, which could spontaneously fold into its native-like structure without additional refolding process or eukaryotic folding factors. The improved IgE-binding activity and simplified preparation method could greatly facilitate it to be a robust allergen material for molecular diagnosis of cat allergy. [ABSTRACT FROM AUTHOR]
- Published
- 2024
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