Increased glycosylation efficiency of recombinant proteins in Escherichia coli by auto-induction

An Engineered Pathway for Production of Terminally Sialylated N-glycoproteins in the Periplasm of Escherichia coli

“…Gal-β-1,4-GlcNAc-specific ECA (Figure 2F lower panel), while the glycosylated FN3 containing the tetrasaccharide glycan (Ding et al, 2017) was strongly detected by ECA ( Figure 2F lower panel) but not by SNA-I ( Figure 2F upper panel). These results indicate the presence of α-2,6-linked sialylation in the glycoengineered FN3.…”

“…pglB, lsgCDEF, GlcNAc-phosphate transferase gene wecA and membrane translocase gene pglK were cloned in the pIG6-compatible plasmid pACYC184 to generate plasmid pC15-plsg with the following format: Arabinose promoter -rbs -pglB -rbs1 -wecA -rbs2-pglK -ompA rbs -lsgCDEF -rrnB terminator. The expression plasmid pIG6-FN3-Gly-1 was previously described for the production of FN3 and glycosylated FN3 proteins (Ding et al, 2017). The sequences of all plasmids were confirmed by DNA sequencing.…”

“…After harvesting of cells by centrifugation, FN3 acceptor proteins were purified using 1 ml HisTrap columns, and desalted using a PD-10 column (Hu et al, 2013). Unglycosylated FN3 and glycosylated FN3 with the tetrasaccharide glycan (Gal-β-1,4-GlcNAc-β -1,3-Gal-β-1,3-GlcNAc-) were produced and purified as previously reported (Ding et al, 2017). Protein concentrations were determined by BCA assay (Pierce).…”

“…(C) Growth curves of E. coli DKK601/pIG6-FN3-Gly-1 (expressing FN3 protein without sialylation pathway) and E. coli DKK601/pIG6-Sia + pC15-plsg (expressing modified FN3 protein with sialylation pathway) cells after induction of recombinant protein expression and glycosylation. Purified unmodified FN3, N-glycosylated FN3 (FN3-Gly) and sialylated FN3 (FN3-Sia) produced over 5 h at 28 • C in E. coli DKK601 cells containing the FN3 gene alone; FN3 and lsgCDEF, wecA, pglB and pglK genes for N-glycosylation (FN3-Gly) (Ding et al, 2017); or additional neuBCA and pl-ST6 genes to effect sialylation (FN3-Sia), detected by (D) Coomassie-stained SDS-PAGE, (E) Western blotting using anti-FLAG M1 antibody, and (F) lectin blotting using Neu5Ac-α-2,6-Gal/GalNAc-specific SNA-I (upper panel) or Gal-β-1,4-GlcNAc-specific ECA (lower panel) lectins. (D-F) lane 1-3: purified FN3, FN3-Gly, FN3-Sia.…”

Genetic engineering approaches to improve posttranslational modification of biopharmaceuticals in different production platforms

“…Bacterial expression systems, mainly Escherichia coli, are widely used for the generation of smaller drugs, including hormones, growth factors, and even more innovative therapeutic agents like peptibodies or peptide fragment crystallizable (Fc) region fusions (Baumgarten, Jimmy Ytterberg, Zubarev, & de Gier, 2018;Shimamoto, Gegg, Boone, & Quéva, 2012). Unlike mammalian cells, bacterial microorganisms lack the ability to perform most PTMs, including glycosylation, which is essential for the production of active biopharmaceuticals (Brown et al, 2017;Ding et al, 2017). In addition, correct protein folding, disulfide bond formation, as well as proper secretion are not a given in most bacterial species leading to the formation of inclusion bodies (Martínez-Alonso, González-Montalbán, García-Fruitós, & Villaverde, 2009).…”