E. coli separatome-based protein expression and purification platform

Patent metadata

Field	Value
Publication number	US-9822371-B2
Application number	US-201415022852-A
Country	US
Kind code	B2
Filing date	Sep 17, 2014
Priority date	Sep 17, 2013
Publication date	Nov 21, 2017
Grant date	Nov 21, 2017

Abstract

Provided is a separatome-based peptide, polypeptide, and protein expression and purification platform based on the juxtaposition of the binding properties of host cell genomic peptides, polypeptides, and proteins with the characteristics and location of the corresponding genes on the host cell chromosome of E. coli . The separatome-based protein expression and purification platform quantitatively describes and identifies priority deletions, modifications, or inhibitions of certain gene products to increase chromatographic separation efficiency, defined as an increase in column capacity, column selectivity, or both, with emphasis on the former. Moreover, the separatome-based protein expression and purification platform provides a computerized knowledge tool that, given separatome data, and a target recombinant peptide, polypeptide, or protein, intuitively suggests strategies facilitating efficient product purification. The separatome-based protein expression and purification platform is an efficient bioseparation system that intertwines host cell expression systems and chromatography.

First claim

What is claimed is:

1. An E. coli wherein a combination of genes selected from the group consisting of the following combinations is deleted:
ΔhldDΔusgΔrraA;
ΔhldDΔusgΔrraAΔcutA;
ΔhldDΔusgΔrraAΔcutAΔnagD;
ΔhldDΔusgΔrraAΔcutAΔnagDΔspeA;
ΔhldDΔusgΔrraAΔcutAΔnagDΔspeAΔgldA;
ΔhldDΔusgΔrraAΔcutAΔnagDΔspeAΔgldAΔglnA;
ΔhldDΔusgΔrraAΔcutAΔnagDΔspeAΔgldAΔglnAΔmetE;
ΔhldDΔusgΔrraAΔcutAΔnagDΔspeAΔgldAΔglnAΔmetEΔtgt;
ΔhldDΔusgΔrraAΔcutAΔnagDΔspeAΔgldAΔglnAΔmetEΔtgtΔargG;
ΔhldDΔusgΔrraAΔcutAΔnagDΔspeAΔgldAΔglnAΔmetEΔtgtΔargGΔtypA;
ΔhldDΔusgΔrraAΔcutAΔnagDΔspeAΔgldAΔglnAΔmetEΔtgtΔargGΔtypAΔentF;
ΔhldDΔusgΔrraAΔcutAΔnagDΔspeAΔgldAΔglnAΔmetEΔtgtΔargGΔtypAΔentFΔycaO;
ΔhldDΔusgΔrraAΔcutAΔnagDΔspeAΔgldAΔglnAΔmetEΔtgtΔargGΔtypAΔentFΔycaOΔslyD;
ΔhldDΔusgΔrraAΔcutAΔnagDΔspeAΔgldAΔglnAΔmetEΔtgtΔargGΔtypAΔentFΔycaOΔslyDΔgatZ;
ΔhldDΔusgΔrraAΔcutAΔnagDΔspeAΔgldAΔglnAΔmetEΔtgtΔargGΔtypAΔentFΔycaOΔslyDΔgatZΔilvB;
ΔhldDΔusgΔrraAΔcutAΔnagDΔspeAΔgldAΔglnAΔmetEΔtgtΔargGΔtypAΔentFΔycaOΔslyDΔgatZΔilvBΔglgP;
ΔhldDΔusgΔrraAΔcutAΔnagDΔspeAΔgldAΔglnAΔmetEΔtgtΔargGΔtypAΔentFΔycaOΔslyDΔgatZΔilvBΔglgPΔnusA; and
ΔhldDΔusgΔrraAΔcutAΔnagDΔspeAΔgldAΔglnAΔmetEΔtgtΔargGΔtypAΔentFΔycaOΔslyDΔgatZΔilvBΔglgPΔnusAΔmet.

2. A method of preparing a pharmaceutical or veterinary composition comprising a therapeutic peptide, polypeptide, or protein, comprising the steps of:
i) expressing said therapeutic peptide, polypeptide, or protein in said E. coli of claim 1 ;
ii) in the case where said therapeutic peptide, polypeptide, or protein is not secreted from said E. coli , preparing a lysate of said E. coli containing said therapeutic peptide, polypeptide, or protein, producing an initial therapeutic peptide-, polypeptide-, or protein-containing mixture; or
iii) in the case where said therapeutic peptide, polypeptide, or protein is secreted from said E. coli , harvesting culture medium in which said E. coli is grown, containing said therapeutic peptide, polypeptide, or protein, thereby obtaining an initial therapeutic peptide-, polypeptide-, or protein-containing mixture;
iv) chromatographing said initial therapeutic peptide-, polypeptide-, or protein-containing mixture of step ii) or step iii) via affinity or adsorption-based, non-affinity chromatography and collecting elution fractions, thereby obtaining one or more fractions containing an enriched amount of said therapeutic peptide, polypeptide, or protein relative to other peptides, polypeptides, or proteins in said fraction compared to the amount of said therapeutic peptide, polypeptide, or protein relative to other peptides, polypeptides, or proteins in said initial protein mixture;
v) optionally, further chromatographing an enriched fraction of step iv) to obtain said therapeutic peptide, polypeptide, or protein in a desired degree of purity; and
vi) recovering said therapeutic peptide, polypeptide, or protein.

3. The method of claim 2 , further comprising formulating said therapeutic peptide, polypeptide, or protein with a pharmaceutically or veterinarily acceptable carrier, diluent, or excipient to produce a pharmaceutical or veterinary composition, respectively.

4. The method of claim 2 , wherein said adsorption-based, non-affinity chromatography is ion exchange chromatography.

5. A method of purifying an enzyme, comprising the steps of:
i) expressing said enzyme in said E. coli host cell of claim 1 ;
ii) in the case where said enzyme is not secreted from said E. coli host cell, preparing a lysate of said E. coli containing said enzyme, producing an initial enzyme-containing mixture; or
iii) in the case where said enzyme is secreted from said E. coli , harvesting culture medium in which said E. coli is grown, containing said enzyme, thereby obtaining an initial enzyme-containing mixture;
iv) chromatographing said initial enzyme-containing mixture of step ii) or step iii) via affinity or adsorption-based, non-affinity chromatography and collecting elution fractions, thereby obtaining one or more fractions containing an enriched amount of said enzyme relative to other peptides, polypeptides, or proteins in said fraction compared to the amount of said enzyme relative to other peptides, polypeptides, or proteins in said initial protein mixture;
v) optionally, further chromatographing an enriched fraction of step iv) to obtain said enzyme in a desired degree of purity; and
vi) recovering purified enzyme.

6. The method of claim 5 , wherein said adsorption-based, non-affinity chromatography is ion exchange chromatography.

Assignees

• Univ Arkansas
• Univ Of Pittsburgh—Of The Commonwealth System Of Higher Education
• Univ Arkansas
• Univ Of Pittsburgh—Of The Commonwealth Systems Of Higher Education

Inventors

• Brune Ellen M
• Beitle Robert R
• Ataai Mohammad M
• Bartlow Patrick R
• Henry Ralph L

Classifications

• C12P21/02

  having a known sequence of two or more amino acids, e.g. glutathione · CPC title

• C12N15/70 Primary

  Vectors or expression systems specially adapted for E. coli · CPC title

• C12N9/00

  Enzymes; Proenzymes; Compositions thereof (preparations containing enzymes for cleaning teeth A61K8/66, A61Q11/00; medicinal preparations containing enzymes or proenzymes A61K38/43; enzyme containing detergent compositions C11D; {enzymes with nucleic acid structure, e.g. ribozymes, C12N15/113}); Processes for preparing, activating, inhibiting, separating or purifying enzymes (preparation of malt C12C1/00) · CPC title

• C12P21/00

  Preparation of peptides or proteins (single cell protein C12N1/00) · CPC title