University of Oulu

Oulun Yliopisto

Lloyd Ruddock

Feras Hatahet

The present invention relates to a method for producing a protein of interest containing one or more disulfide bonds in its native state. The method comprises that a prokaryotic host cell is genetically engineered to express the protein of interest and a sulfhydryl oxidase in the cytoplasm of the host cell. The protein of interest is formed in a soluble form and contains disulfide bonds due to the presence of the sulfhydryl oxidase in the cytoplasm of said host cell. The present invention relates also to a prokaryotic host cell and a vector system for producing a protein of interest containing natively folded disulfide bonds.

Method for producing natively folded proteins in a prokaryotic host

The present invention relates to a method for producing natively folded disulfide bond containing proteins in a prokaryotic host. The method comprises that in the cytoplasm of a prokaryotic cell is expressed protein(s) of interest that naturally contain disulfide bonds and naturally occurring or inverted transmembrane enzyme, wherein the cysteines of the active site(s) are naturally or after genetic engineering located towards the prokaryotic cytoplasm. The enzyme is selected from the group of VKOR, inverted VKOR (iVKOR) and inverted Dsb B (iDsb B). In the prokaryotic cell is also expressed cytoplasmic DsbA or a corresponding protein being capable of providing electrons to the active site(s) of VKOR, iVKOR or iDsbB. The invention relates also to a prokaryotic host cell and a vector system for producing natively folded disulfide bond containing proteins.

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Oulu, Finland

Lloyd Ruddock