Phenyl sepharose fast flow

One ml of the sample was diluted 20 times in 1.7 M Ammonium Sulfate buffer and then loaded on to the Hydrophobic interaction column column (1 ml Phenyl Sepharose Fast Flow, low substitution, from GE) at 1 ml/min. Fractions were collected every 0.5 ml. An AKTA UPC 10 FPLC system from GE at 4° was used for all chromatography steps.

The SARS-CoV-2 RBD gene (GenBank accession number MN908947.3) was synthesized, ligated into the pPICZαA vector, and subsequently transferred into glycoengineered P. Pastoris cells and RBD-wild type protein (RBD-WT) was successfully expressed and purified (Liu et al., 2022 (link)). The gene sequence of K417N, E484K, and N501Y sites were mutated, and the target gene was cloned to generate expression vector pPICZαA-RBD-Beta. BglII-linearized pPICZαA-RBD-Beta was transferred into glycoengineered P. pastoris cells by electric shock, and expression of the target protein was induced by addition of methanol. Positive clones were screened by SDS-PAGE and western blot. The primary antibody was anti-RBD-WT rabbit serum (produced and kept in our laboratory) and the secondary antibody was goat anti-rabbit-HRP (1:2500, SAB3700885, SIGMA).
The positive strain highly expressing RBD-Beta was fermented in a 5 L biofermenter, and the target protein was induced with methanol for expression. The supernatant was passed through the following chromatographic columns: Capto MMC, Phenyl Sepharose Fast Flow, Source 30Q, Source 30S, and Superdex-G75 (all from GE Healthcare, Cal., United States) to obtain high purity RBD-Beta protein.