Production of soluble recombinant protein without n-terminal methionine
Inventors
OGANESYAN, Natalia • Lees, Andrew • Chang, Min-Ju
Assignees
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Publication Number
US-11060123-B2
Publication Date
2021-07-13
Expiration Date
Abstract
The present invention is directed to the cells, compositions and methods for the production of recombinant protein, wherein an f-met group on the 5′-terminus is enzymatically removed. In particular, the invention is directed to a production process for obtaining high levels of soluble recombinant CRM197 protein from E. coli. Cells preferably contain one or more mutations of disulfide reductase genes, so that disulfide reductase activity is reduced. The invention also relates to purification method for CRM197 as well as characterization of properly folded CRM197 protein.
Core Innovation
The invention relates to engineering a recombinant cell to produce a protein containing one or more sulfide linkages with a 5′ terminus that initially contains a methionine and is then processed so that the methionine is removed from the 5′ terminus by a peptidase. The method further includes isolating the protein after the peptidase removes the methionine from the 5′ terminus of the protein expressed.
The recombinant cell is characterized by reduced activity of one or more disulfide reductase enzymes, and the protein is expressed from a recombinant cell containing a genome and an expression vector that encodes the protein sequence. In the provided description, recombinant E. coli strains are engineered to shift redox toward an oxidative state by reducing disulfide reductase activity so that soluble cytoplasmic disulfide-bond proteins can be produced.
To enable removal of the 5′ methionine, a peptidase is expressed from a gene of the recombinant cell to remove the methionine from the 5′ terminus of the protein expressed. The description further indicates that methionine aminopeptidase (MAP/METAP1/METAP2) can be used, and that the resulting protein can be confirmed, including examples describing MS confirmation of complete f-Met cleavage for CRM197.
Claims Coverage
The claims include one independent claim covering a method that combines reduced disulfide reductase activity, expression of a protein with a 5′ terminus containing methionine, expression of a peptidase that removes the 5′ methionine, and isolation of the resulting protein. Dependent claims further refine the method with specific peptidase and isolation constraints and defined protein target examples.
Reduced disulfide reductase activity for sulfide-linked protein production
The recombinant cell expresses the protein sequence while the recombinant cell has reduced activity of one or more disulfide reductase enzymes, enabling production of a protein containing one or more sulfide linkages.
5′ methionine-containing 5′ terminus during expression
The protein expressed from the recombinant cell has a 5′ terminus that contains a methionine.
Peptidase removes 5′ methionine from the expressed protein
The method includes expressing a peptidase from a gene of the recombinant cell, where the peptidase removes the methionine from the 5′ terminus of the protein expressed.
Isolating the processed protein
After peptidase processing, the method includes isolating the protein.
Methionine aminopeptidase peptidase
In a defined refinement, the peptidase is a methionine aminopeptidase.
Chromatography isolation
In a defined refinement, the isolating step is performed using chromatography.
Overall, claim coverage centers on producing sulfide-linked proteins in a recombinant cell engineered for reduced disulfide reductase activity so the expressed protein initially has a 5′ methionine, followed by expressing a peptidase that removes that 5′ methionine and isolating the processed protein, with refinements that specify methionine aminopeptidase and chromatography isolation.
Stated Advantages
Not explicitly described in patent.
Documented Applications
Not explicitly described in patent.
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