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Publication Number
US-12410393-B2
Publication Date
2025-09-09
Expiration Date
Abstract
This disclosure provides an E. coli strain, which lacks thioredoxin reductase activity encoded by trxB and thioredoxin 1 activity encoded by trxA, and glutathione reductase activity encoded by gor. Said E. coli strain expresses a mutated AhpC protein having glutathione reductase activity and a cytosolic prokaryotic disulfide isomerase. The E. coli strain has an oxidative cytosol and can be used to efficiently produce proteins having disulfide bonds.
Core Innovation
The invention provides an E. coli platform having an oxidizing cytosol in which trxB, trxA, and gor mutations remove the corresponding activities, while a mutated AhpC protein provides glutathione reductase activity. Cytosolic DsbC supports disulfide-bond folding. The disclosure additionally describes soluble recombinant protein expression using the genetically modified strain and associated strain-containing kits.
The reported screening result describes the Snuggle strain with an additional trxA inactivation compared with Shuffle, producing higher levels of challenging antibody light chains. SDS-PAGE indicates improved soluble expression for anti-MUC1 and anti-CD74 light chains, and four light chains show 20–110% higher relative production than in Shuffle. Trastuzumab light chain is soluble across tested strains.
Claims Coverage
The claims contain two independent claims, covering two inventive features directed to an engineered E. coli strain and a method for expressing soluble recombinant proteins.
E. coli strain with specified mutations and mutated AhpC
Claim 1 covers an E. coli strain that lacks thioredoxin reductase activity due to genetic mutation of trxB, lacks thioredoxin 1 activity due to genetic mutation of trxA, and lacks glutathione reductase activity due to genetic mutation of gor. The strain expresses a mutated AhpC protein with glutathione reductase activity.
Expression of soluble recombinant proteins in E. coli
Claim 13 covers expressing a protein of interest as a soluble recombinant protein in an E. coli strain with an oxidizing cytosol. The strain has genetic mutations of trxB, trxA, and gor that eliminate the corresponding activities and expresses a mutated AhpC protein with glutathione reductase activity.
The independent claims cover the specified E. coli strain and its use for soluble recombinant protein expression. Both claims center on mutations affecting TrxB, TrxA, and Gor activity and expression of mutated AhpC with glutathione reductase activity.
Stated Advantages
The Snuggle strain shows improved soluble expression of anti-MUC1 and anti-CD74 antibody light chains compared with Shuffle.
Four antibody light chains show 20–110% higher relative production in Snuggle than in Shuffle.
Documented Applications
Expression of soluble recombinant proteins of interest in E. coli.
Expression of antibody light chains, including anti-MUC1, anti-CD74, and trastuzumab light chains.
Kits containing the E. coli strain and a growth medium.
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