Method for producing natively folded proteins in a prokaryotic host
AssigneeUniversity of Oulu
InventorLloyd Ruddock
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Abstract
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.
Claims13
A prokaryotic host cell comprising one or more expression vectors encoding a cytoplasmically expressing sulfhydryl oxidase catalyzing a reaction:
- dithiol+O2→2disulfides+2H2O, and a cytoplasmically expressing eukaryotic or prokaryotic protein of interest containing one or more disulfide bonds wherein the protein of interest is produced in a soluble form and containing natively folded disulfide bonds in the cytoplasm of said prokaryotic host cell due to presence of the sulfhydryl oxidase in the cytoplasm.
A vector system for a cytoplasmic expression in a prokaryotic host cell of a recombinant eukaryotic or prokaryotic protein of interest in a soluble form containing natively folded disulfide bonds, said vector system comprising:
- a vector comprising a nucleotide sequence or having a site for the nucleotide sequence encoding a eukaryotic or a prokaryotic cytoplasmically expressing protein of interest said protein containing one or more disulfide bonds, and a nucleotide sequence encoding cytoplasmically expressing sulfhydryl oxidase catalyzing reaction dithiol+O2→2 disulfides+2 H2O for folding the disulfide bonds for production of the eukaryotic or prokaryotic cytoplasmically expressing protein of interest in soluble form containing natively folded disulfide bonds, or
- a first vector comprising a nucleotide sequence encoding a eukaryotic or a prokaryotic cytoplasmically expressing protein of interest, or having a site for a nucleic acid sequence encoding a eukaryotic or a prokaryotic cytoplasmically expressing protein of interest, and
- a second vector comprising a nucleotide sequence encoding a cytoplasmically expressing sulfhydryl oxidase catalyzing reaction dithiol+O2→2 disulfides+2 H2O, for folding the disulfide bonds for production of the eukaryotic or prokaryotic cytoplasmically expressing protein of interest in soluble form containing natively folded disulfide bonds,
- and regulatory regions for multiplying and expressing said nucleotide sequences in a prokaryotic host cell.
Dependencies are inferred from the claim text.