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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).
Combination of engineering the substrate and Ca2+ binding domains of heparinase I to improve the catalytic activity
Published in Preparative Biochemistry & Biotechnology, 2023
Hua-Ping Zhou, Ding-Ran Wang, Chen-Lu Xu, Ye-Wang Zhang
The low activity of heparinase I was one of the main causes that currently hamper its industrial application. Therefore, structural engineering of heparinase I is an effective approach to improve the catalytic activity of heparinase I. The enzyme production reached 20,650 U·L−1 by fuzing maltose binding protein (MBP) with heparinase I to form a fusion protein.[24] After mutation of C297 on MBP-Hep I, its enzyme activity (149 U/mg) was increased by 30.6% by inhibiting the formation of intramolecular disulfide bonds.[25] The clone E. coli-heparinase-I133T/P316T was obtained by directed evolution of heparinase I, and its enzymatic activity was increased by 57.8%.[26] The amino acid residues near the active pocket of heparinase I were modified by protein engineering. The multiple-point mutant S169D/A259D showed a 122.05% increase in enzyme activity.[12] However, the present heparinase I is not enough for its industrial application. Therefore, we need to find new candidate mutants to further improve the catalytic performance of heparinase I.
Preparation and self-cleavage of fusion soluble farnesyl diphosphate synthase in E. coli
Published in Preparative Biochemistry & Biotechnology, 2023
Wenfeng Ni, Zixuan Wang, Aifang Zheng, Ying Zhao
To further enhance FPPS solubility, multiple fusion tags (DsbA, ZZ, and GB1) were used. Specifically, the recombinant plasmids, pDsbA-ispA, pZZ-ispA, and pGB1-ispA (Figure 4), were transformed into E. coli BL21 (DE3). After cell growth and induction, SDS-PAGE of the cell lysates revealed that the fusion proteins (DsbA-FPPS, ZZ-FPPS, and GB1-FPPS) were successfully expressed in E. coli BL21 (DE3). The molecular weights of the recombinant fusion FPPS were approximately 58, 50, and 43 kDa, respectively, which was consistent with the predicted molecular weights. Most of the DsbA-FPPS, ZZ-FPPS, and GB1-FPPS could be accumulated in a soluble form in the LB medium, suggesting that the three tags solubilize FPPS to a certain degree. GB1-FPPS exhibited the highest protein soluble yields expressed in E. coli BL21 (DE3), which was 5.1 and 1.4 times of DsbA-FPPS and ZZ-FPPS, respectively (Table 1). Additionally, compared with other tags, such as Maltose Binding Protein (MBP),[34] GB1 with a smaller molecular weight may have a weaker impact on the structure of the target protein after connecting to its terminus, which has certain potential applications in production. Therefore, the most suitable fusion tag for soluble FPPS expression is GB1.
Cloning, expression, and characterization of a novel heparinase I from Bacteroides eggerthii
Published in Preparative Biochemistry & Biotechnology, 2020
Cai-Yun Liu, Wen-Bin Su, Li-Bin Guo, Ye-Wang Zhang
To solve this problem, fusion expression with a tag was used to help the folding of the target protein. A serious of fusion tags like cellulose-binding domain (CBD), glutathione-S-transferase (GST) and maltose-binding protein (MBP) were applied to produce soluble Hep I in E. coli. But the recombinant CBD-Hep I was still expressed mainly in inactive inclusion bodies,[19] while GST-Hep I had to be expressed at a low temperature to achieve a higher solubility.[13] Approximately 90% of the soluble protein was produced when Hep I was fused to MBP in E. coli at 15 °C. However, the Km of the MBP-Hep I was 4.8 times higher than its native form from F. heparinum, indicating that the affinity of MBP-Hep I toward its substrate heparin might have been hindered to some extent by the MBP tag.[20]