Improved production of Humira antibody in the genetically engineered Escherichia coli SHuffle, by co-expression of human PDI-GPx7 fusions

Abstract

Microbial production of antibodies offers the promise of cheap, fast, and efficient production of antibodies at an industrial scale. Limiting this capacity in prokaryotes is the absence of the post-translational machinery, present in dedicated antibody producing eukaryotic cell lines, such as B cells. There has been few and limited success in producing full-length, correctly folded, and assembled IgG in the cytoplasm of prokaryotic cell lines. One such success was achieved by utilizing the genetically engineered Escherichia coli strain SHuffle with an oxidative cytoplasm. Due to the genetic disruption of reductive pathways, SHuffle cells are under constant oxidative stress, including increased levels of hydrogen peroxide (H2O2). The oxidizing capacity of H2O2 was linked to improved disulfide bond formation, by expressing a fusion of two endoplasmic reticulum-resident proteins, the thiol peroxidase GPx7 and the protein disulfide isomerase, PDI. In concert, these proteins mediate disulfide transfer from H2O2 to target proteins via PDI-Gpx7 fusions. The potential of this new strain was tested with Humira, a blockbuster antibody usually produced in eukaryotic cells. Expression results demonstrate that the new engineered SHuffle strain (SHuffle2) could produce Humira IgG four-fold better than the parental strain, both in shake-flask and in high-density fermentation. These preliminary studies guide the field in genetically engineering eukaryotic redox pathways in prokaryotes for the production of complex macromolecules. Key points • A eukaryotic redox pathway was engineered into the E. coli strain SHuffle in order to improve the yield of the blockbuster antibody Humira. • The best peroxidase-PDI fusion was selected using bioinformatics and in vivo studies. • Improved yields of Humira were demonstrated at shake-flask and high-density fermenters.

Full-length recombinant antibodies from Escherichia coli : production, characterization, effector function (Fc) engineering, and clinical evaluation

“… 104 Expression of adalimumab was increased 4-fold in both shake-flask and high cell density fermentation by overexpressing a fusion of the human protein disulfide isomerase (PDI) and peroxidase 7 genes (PDI-GPx7 fusion), combined with the oxidizing power of H 2 O 2 accumulated in SHuffle® cells 105 due to the inability of the mutated ahp C gene to reduce H 2 O 2 . 106 In shake-flask cultures, the final adalimumab titer was 168 mg/L vs. 427 mg/L for SHuffle® and SHuffle2® hosts, respectively. In fermentors, adalimumab titer was 137 ng/mL/OD 600 cells vs. 475 ng/mL/OD 600 cells for SHuffle® and SHuffle2® hosts, respectively.…”

Research progress of glutathione peroxidase family (GPX) in redoxidation

“…Glutathione peroxidase 7 (GPX7) is a non-selenocysteine active neutral antioxidant enzyme in mammals, also known as non-selenium cysteine phGPX (NPGPX) due to its homology to phospholipid peroxide GPX (phGPX = GPX4) (Chen et al, 2016). However, unlike GPX4, GPX7 does not contain a GSH-binding domain, and when expressed and purified in E. coli, there is little GPX activity (Lénon et al, 2020). The mammalian GPX7 gene contains 3 exons, encodes 187 amino acids, and has a predicted molecular weight of 22 kDa.…”

Can thiol-based redox systems be utilized as parts for synthetic biology applications?

“…Arguably, the most comprehensive redox synthetic biology circuits are those that aimed to facilitate cytoplasmic disulfide bond formation in E. coli which led to the Origami (Novagen), SHuffle (NEB) [ 175 ] and SHuffle2 strains [ 176 ]. In these cells, the functional separation and redundancy between the NADPH and NADH-dependent thiol-based reactions (cf.…”

“…Figure 1 ), and a mutant peroxidase (AhpC*) reduced glutathionylated glutaredoxin and GSH [ 92 ] to support metabolic pathways such as ribonucleotide reductase (Nrd) cycling. A PDI-GPx7 chimera was used to reduce hydrogen peroxide and, together with thioredoxin, oxidize protein thiols, while disulfide isomerization by DsbC was used to enable correctly folding of a target antibody [ 176 ]. The hypothetical distribution between the oxidized (pink) and reduced (blue) moieties are shown for each redox couple.…”