Por favor, use este identificador para citar o enlazar este ítem: https://hdl.handle.net/20.500.12008/55709

Título:

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

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Autor:

Lénon, Marine
Ke, Na
Szady, Cecily
Sakhtah, Hassan
Ren, Guoping
Manta, Bruno
Causey, Bryce
Berkmen, Mehmet

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Tipo de documento:

Artículo

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Palabras clave:

Disulfide bond
E. coli
GPx7
Humira production
PDI
Redox engineering
SHuffle

Descriptores:

ADALIMUMAB
PROTEÍNAS DE ESCHERICHIA COLI
ESCHERICHIA COLI
GENÉTICA
GLUTATIÓN PEROXIDASA
HUMANOS
PERÓXIDO DE HIDRÓGENO
PEROXIDASAS
PROTEIN DISULFIDE-ISOMERASES

Año de publicación:

2020

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Resumen:

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.

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Springer International

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Applied Microbiology and Biotechnology. 2020;104(22):9693-9706

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Citación:

Lénon M, Ke N, Szady C y otros. Improved production of Humira antibody in the genetically engineered Escherichia coli SHuffle, by co-expression of human PDI-GPx7 fusions. Applied Microbiology and Biotechnology [en línea]. 2020;104(22):9693-9706

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Licencia Creative Commons Atribución (CC - By 4.0)

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