Methods

Protein Purification and Crystallization

To determine the binding structure of the spike protein G3P[10], a series of purification and crystallization techniques were employed. The His-Tag VP8 protein was initially crystallized using the hanging drop vapor diffusion method, but substrate binding was not successful. To enhance binding efficiency, GST-tag affinity purification methods were employed instead. 

Affinity Chromatography Purification:

1. His-Tag Purification: The VP8 protein was first purified using a nickel-nitrilotriacetic acid (Ni-NTA) column, which specifically binds to histidine residues.

Due to low yield from His-tag cleavage, the His-tag was replaced with a GST-tag. This modification aimed to enhance protein purification and facilitate better binding to the substrate.

2. GST-Tag Purification: For further purification, a glutathione Sepharose column was used to selectively bind the GST-tagged protein.

Affinity Tag Removal: To remove GST-tag, thrombin protease was employed. Thrombin recognizes and cleaves specifically at the recognition site located immediately after GST tag, leaving the VP8 protein intact for further analysis.

SDS-PAGE Gel Analysis: the presence or absence of the tag after cleavage was assessed by comparing the molecular weight of the cleaved protein with that of the expected untagged protein. The gel was stained with Coomassie Brilliant Blue, and the bands were analyzed to confirm the successful removal of the tag, with the target protein migrating at the expected size corresponding to the cleaved product.

Protein Concentration and Crystallization Trials: Following tag removal, the protein was centrifuged and concentrated to ensure sufficient quantities for crystallization. Protein concentration was measured using a Bradford assay to verify purity and concentration before proceeding with crystallization trials.

This purification and crystallization strategy was designed to obtain untagged VP8 protein suitable for structural and binding analyses.