The AMP deaminase of the mollusk Helix pomatia is an unexpected member of the adenosine deaminase-related growth factor (ADGF) family.

We report here the first occurrence of an adenosine deaminase-related growth factor (ADGF) that deaminates adenosine 5' monophosphate (AMP) in preference to adenosine. The ADGFs are a group of secreted deaminases found throughout the animal kingdom that affect the extracellular concentration of adenosine by converting it to inosine. The AMP deaminase studied here was first isolated and biochemically characterized from the roman snail Helix pomatia in 1983. Determination of the amino acid sequence of the AMP deaminase enabled sequence comparisons to protein databases and revealed it as a member of the ADGF family. Cloning and expression of its cDNA in Pichia pastoris allowed the comparison of the biochemical characteristics of the native and recombinant forms of the enzyme and confirmed they correspond to the previously reported activity. Uncharacteristically, the H. pomatia AMP deaminase was determined to be dissimilar to the AMP deaminase family by sequence comparison while demonstrating similarity to the ADGFs despite having AMP as its preferred substrate rather than adenosine.

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

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.