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Use of Enzymes in the Downstream Processing of Biopharmaceuticals
Published in Peter Grunwald, Pharmaceutical Biocatalysis, 2019
A specific application where enzymes are used to modify the structure of a precursor of the target molecule is the removal of affinity tags (Arnau et al., 2006; Waugh, 2011). These tags, which are usually composed of a number of exogenous amino acid sequences, are added by inserting the corresponding coding sequences next to those that code for the protein of interest. Tags that have a high affinity for a specific ligand like the common hexa-histidine (HHHHHH) tag (Hoffmann and Roeder, 1991) or that contain an epitope recognized by immobilized antibodies like the FLAG (DYKDDDDK) (Hopp et al., 1988; Knappik and Pluckthun, 1994) and Streptag II (WSHPQFEK) (Skerra and Schmidt, 2000; Schmidt and Skerra, 2007) tags, are commonly used to facilitate purification. The high affinity of the corresponding tag-protein fusions to their ligands makes it possible to purify them by affinity chromatography with immobilized nickel (His tag), antibody (FLAG tag) and streptavidin (Streptag II) matrices (Arnau et al., 2006; Unal et al., 2008). In other cases, tags like glutathione S-transferase (GST) (Ren et al., 2003), maltose binding protein (MBP) (Guan et al., 1988) and elastin-like polypeptides (ELP) (Kowalczyk et al., 2014) are introduced to improve the solubility of the target protein. Proteins fused with these tags are also easier to purify due to their ability to bind to specific matrices (e.g., glutathione–Sepharose for GST, amylose for MBP) or to undergo temperature-induced aggregation (ELPs) (Arnau et al., 2006).
Expression and functional identification of recombinant SARS-CoV-2 receptor binding domain (RBD) from E. coli system
Published in Preparative Biochemistry & Biotechnology, 2022
Xiangzheng Gao, Shanshan Peng, Shengsheng Mei, Keying Liang, Muhammad Saleem Iqbal Khan, Eu Gene Vong, Jinbiao Zhan
The mouse anti-6 × His tag monoclonal antibody and mouse anti-Flag tag monoclonal antibody were obtained from Proteintech Group (USA). The rabbit anti- SARS-CoV-2 RBD polyclonal antibody (pAb) was purchased from ABclonal Tech (Wuhan, China). The goat anti-mouse IgG-Alexa Fluor® 647 antibody was obtained from HuaBio biotech (Hangzhou, China). The HRP-conjugated goat anti-mouse IgG and HRP-conjugated goat anti-rabbit IgG antibodies were obtained from BBI Life Sciences (Shanghai, China). The NcmECL Ultra Kit and Trypsin solution were purchased from NCM Biotech (Suzhou, China).