Isoelectric point determination by imaged CIEF of commercially available SARS‐CoV‐2 proteins and the hACE2 receptor

Krebs, Finja; Scheller, Christin; Grove‐Heike, Kristina; Pohl, Lena; Wätzig, Hermann

In order to contribute to the scientific research on the severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2), we have investigated the isoelectric points (pI) of several related proteins, which are commercially available: the receptor‐binding domain (RBD) with His‐ and Fc‐tag, the S1 subunit with His‐tag, the S1/S2 subunits with His‐tag and the human angiotensin‐converting enzyme 2 (hACE2) with His‐tag. First, the theoretical pI values, based on the amino acid (AA) sequences of the proteins, were calculated using the ProtParam tool from the Bioinformatics Resource Portal ExPASy. The proteins were then measured with the Maurice imaged CIEF system (native fluorescence detection), testing various measurement conditions, such as different ampholytes or ampholyte mixtures. Due to isoforms, we get sections with several peaks and not just one peak for each protein. The determined pI range for the RBD/Fc is 8.24–9.32 (theoretical pI: 8.55), for the RBD/His it is 7.36–9.88 (8.91) and for the S1/His it is 7.30–8.37 (7.80). The pI range of the S1/S2/His is 4.41–5.87 (no theoretical pI, AA sequence unknown) and for hACE2/His, the determined global range is 5.19–6.11 (5.60) for all experimental conditions chosen. All theoretically derived values were found within these ranges, usually close to the center. Therefore, we consider theoretical values as useful to make predictions about the isoelectric points of SARS‐CoV‐2 proteins. The experimental conditions had only a minor influence on the pI ranges obtained and mainly influenced the peak shapes.