Spike Protein Production in TunaCHO

Various versions of SARS-CoV2 Spike proteins were produced in TunaCHO 7-day process and purified by IMAC. Data shown below are SDS-PAGE images after initial purification. The full length ectodomain versions of Spike protein expressed at around 30 mg/L in TunaCHO and formed trimers based on SEC-HPLC, as expected. There is no evidence of monomer formed.

The furin cleavage site on spike protein is functional, therefore the S protein is cut by furin in CHO cells into two fragments: S1 and S2. Once the furin cleavage site is mutated, the proteins are no longer cleavable. Interestingly S1 and S2 appear to associate after furin cleavage, suggesting strong interaction between S1 and S2 or between S1 and S1. 

Full length spike protein has theoretical molecular weight 138 kD. The protein has many glycosylation sites, the purified protein appears to run at approximately 200 kD in SDS-PAGE. S1 appears at 120 kD and S2 at 80 kD. 

We have obtained binding data with ELISA and Octet which both showed that full length Spike proteins with prefusion stablized conformation bind to ACE2 strongly. This binding is substantially stronger than the interaction between RBD and ACE2 due to avidity of trimers. 

Wild type with furin site removed  (Ref. 46327)

Prefusion conformation with furin site intact  (Ref. 46026)

Prefusion conformation with furin site removed  (Ref. 46328)