Methods and compositions for improved expression of recombinant proteins
Inventors
Blattner, Frederick R. • Novy, Robert E. • Frisch, David A. • Landry, Charles • Choi, Hyunsic • STEFFEN, Eric A. • Brandon, John
Assignees
Interested in licensing this patent?
MTEC can help explore whether this patent might be available for licensing for your application.
Publication Number
US-11667944-B2
Publication Date
2023-06-06
Expiration Date
Abstract
Provided herein are methods for the improved production of periplasmic-targeted recombinant proteins in E. coli host strains. Also provided are E. coli host strains with improved capacity for producing recombinant proteins.
Core Innovation
The invention relates to a method for extracting a recombinant polypeptide from the periplasm of an Escherichia coli host cell. The method uses a first nucleotide sequence encoding the recombinant polypeptide fused to a signal sequence that directs the recombinant polypeptide to the periplasm, with the first nucleotide sequence operably linked to an expression control sequence comprising an inducible promoter. The first and second nucleotide sequences are different and are each linked to an inducible promoter.
A second nucleotide sequence encodes an E. coli phospholipase, with the second nucleotide sequence operably linked to a heterologous expression control sequence comprising an inducible promoter. The host cell is cultured in a medium under conditions permitting co-expression of the recombinant polypeptide and the phospholipase. After co-expression, the cells are contacted with an extraction buffer comprising a chelating agent for at least 6 hours and less than 24 hours at a temperature between 20°C and 30°C to release the recombinant polypeptide from the periplasm into the extraction buffer.
The documented work further supports improved production and recovery by using named recombinant polypeptides, including CRM197, Protein D/PD, Exoprotein A/EPA, and also single-chain antibodies scFv 75127 and scFab YMF10. The disclosed approach includes continuous fermentation support for reduced-genome E. coli strains, including genome deletions and deletion or lack of defined genes such as toxin-antitoxin genes and/or rnc (RNase III). The extraction and recovery are presented as improved periplasm-to-medium extraction efficiencies with direct purification from clarified medium.
Claims Coverage
The independent claim (one inventive concept) covers extraction of a periplasm-targeted recombinant polypeptide from an Escherichia coli host by co-expressing a heterologous E. coli phospholipase from a different inducible promoter and using a chelating-agent extraction buffer for a defined contact time and temperature. Dependent claims further refine the recombinant polypeptide, specific buffer/efficiency constraints, and host strain/genetic constraints, including continuous fermentation mode and genomic engineering limitations.
Periplasm targeting with inducible expression of recombinant polypeptide
A method in which a first nucleotide sequence encodes a recombinant polypeptide fused to a signal sequence directing transfer to the periplasm, where the first nucleotide sequence is operably linked to an inducible promoter expression control sequence.
Inducible expression of an E. coli phospholipase from a different sequence
A method in which a second nucleotide sequence encodes an E. coli phospholipase, where the second nucleotide sequence is operably linked to a heterologous expression control sequence comprising an inducible promoter, with the first and second nucleotide sequences being different.
Chelating-agent extraction of the periplasm-released polypeptide
A method comprising culturing the host to permit co-expression, contacting the E. coli host cells with an extraction buffer comprising a chelating agent for at least 6 hours and less than 24 hours at a temperature between 20°C and 30°C to release the recombinant polypeptide from the periplasm into the extraction buffer, and collecting the recombinant polypeptide from the extraction buffer.
Quantified extraction buffer composition and pH (dependent refinement)
A method in which the extraction buffer in step (b) contains EDTA at 3–7 mM, Tris at 100–200 mM, and has a pH of 7–8.
Quantified extraction efficiency threshold (dependent refinement)
A method in which the extraction extracts at least 80% of the recombinant polypeptide from the periplasm of the E. coli host cell.
Continuous fermentation production constraint (dependent refinement)
A method further culturing the E. coli host cell in continuous fermentation mode for at least 14 days to produce at least 2 grams per liter per day of recombinant protein.
Selected recombinant polypeptide set (dependent refinement)
A method in which the recombinant polypeptide is selected from CRM197, Protein D, or Exoprotein A.
Reduced-genome / rnc gene absence constraint (dependent refinement)
A method performed using an E. coli host cell that lacks a functional rnc gene.
Overall, the claims are centered on extracting a periplasm-targeted recombinant polypeptide by co-expressing an E. coli phospholipase from a different inducible promoter and performing chelating-agent extraction under defined contact time and temperature. Dependent claims further narrow the recombinant polypeptide set, add quantified buffer and extraction-efficiency constraints, impose continuous fermentation production constraints, and specify host genetic limitations including lack of a functional rnc gene.
Stated Advantages
Improved release/extraction of periplasm-targeted recombinant proteins into the extraction buffer upon phospholipase co-expression, as supported by the described experimental results.
Improved periplasm-to-medium extraction efficiencies and recovery during continuous fermentation-supported production.
Simplification enabling direct purification from clarified medium supported by continuous fermentation of reduced-genome E. coli strains.
Documented Applications
Production and extraction/recovery of periplasm-targeted recombinant proteins in Escherichia coli, including CRM197, Protein D/PD, Exoprotein A/EPA, and single-chain antibodies scFv 75127 and scFab YMF10.
Continuous fermentation-supported production of reduced-genome E. coli strains, with deletion or lack of defined genes such as toxin-antitoxin genes and/or rnc, enabling long-term production and direct purification from clarified medium.
Interested in licensing this patent?