Artificial intelligence-driven design of the assembled major cat allergen Fel d 1 to improve its spatial folding and IgE-reactivity

Background: 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. Methods: 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). Results: Among several linkers, 2 x GGGGS got the highest scores, with an overall quality factor of 100. The error value of the residues around the junction of 2 x 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. Conclusion: 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 IgEbinding activity and simplified preparation method could greatly facilitate it to be a robust allergen material for molecular diagnosis of cat allergy.