Patent
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.
1. A method of purifying a target peptide, polypeptide, or protein, comprising the steps of:
1) recombinantly or non-recombinantly expressing said target peptide, polypeptide, or protein in E. coli wherein a combination of genes selected from the group consisting of the following combinations of genes is deleted:
ΔhldDΔusgΔrraA;
ΔhldDΔusgΔrraAΔcutA;
ΔhldDΔusgΔrraAΔcutAΔnagD;
ΔhldDΔusgΔrraAΔcutAΔnagDΔspeA;
ΔhldDΔusgΔrraAΔcutAΔnagDΔspeAΔgldA;
ΔhldDΔusgΔrraAΔcutAΔnagDΔspeAΔgldAΔglnA;
ΔhldDΔusgΔrraAΔcutAΔnagDΔspeAΔgldAΔglnAΔmetE;
ΔhldDΔusgΔrraAΔcutAΔnagDΔspeAΔgldAΔglnAΔmetEΔtgt;
ΔhldDΔusgΔrraAΔcutAΔnagDΔspeAΔgldAΔglnAΔmetEΔtgtΔargG;
ΔhldDΔusgΔrraAΔcutAΔnagDΔspeAΔgldAΔglnAΔmetEΔtgtΔargGΔtypA;
ΔhldDΔusgΔrraAΔcutAΔnagDΔspeAΔgldAΔglnAΔmetEΔtgtΔargGΔtypAΔentF;
ΔhldDΔusgΔrraAΔcutAΔnagDΔspeAΔgldAΔglnAΔmetEΔtgtΔargGΔtypAΔentFΔycaO;
ΔhldDΔusgΔrraAΔcutAΔnagDΔspeAΔgldAΔglnAΔmetEΔtgtΔargGΔtypAΔentFΔycaOΔslyD;
ΔhldDΔusgΔrraAΔcutAΔnagDΔspeAΔgldAΔglnAΔmetEΔtgtΔargGΔtypAΔentFΔycaOΔslyDΔgatZ;
ΔhldDΔusgΔrraAΔcutAΔnagDΔspeAΔgldAΔglnAΔmetEΔtgtΔargGΔtypAΔentFΔycaOΔslyDΔgatZΔilvB;
ΔhldDΔusgΔrraAΔcutAΔnagDΔspeAΔgldAΔglnAΔmetEΔtgtΔargGΔtypAΔentFΔycaOΔslyDΔgat ZΔilvBΔglgP;
ΔhldDΔusgΔrraAΔcutAΔnagDΔspeAΔgldAΔglnAΔmetEΔtgtΔargGΔtypAΔentFΔycaOΔslyDΔgat ZΔilvBΔglgPΔnusA; and
ΔhldDΔusgΔrraAΔcutAΔnagDΔspeAΔgldAΔglnAΔmetEΔtgtΔargGΔtypAΔentFΔycaOΔslyDΔgat ZΔilvBΔglgPΔnusAΔmetH;
2) in the case where said expressed target peptide, polypeptide, or protein is not secreted from said E. coli , preparing a lysate of said E. coli containing said expressed target peptide, polypeptide, or protein, producing an initial expressed target peptide-, polypeptide-, or protein-containing mixture; or
3) in the case where said expressed target peptide, polypeptide, or protein is secreted from said E. coli , harvesting culture medium in which said E. coli is grown, containing said expressed target peptide, polypeptide, or protein, thereby obtaining an initial expressed target peptide-, polypeptide-, or protein-containing mixture;
4) chromatographing said initial expressed target peptide-, polypeptide-, or protein-containing mixture of step 2) or step 3) using an affinity chromatography column comprising an affinity ligand bound to a solid phase, or an adsorption-based, non-affinity chromatography medium-containing column, and collecting elution fractions, thereby obtaining one or more fractions containing an enriched amount of said expressed target peptide, polypeptide, or protein relative to other peptides, polypeptides, or proteins in said fraction compared to the amount of said expressed target peptide, polypeptide, or protein relative to other peptides, polypeptides, or proteins in said initial expressed target peptide-, polypeptide-, or protein-containing mixture; and
5) further chromatographing an enriched fraction of step 4) to obtain said expressed target peptide, polypeptide, or protein in a desired degree of purity.
2. The method of claim 1 , further comprising recovering purified expressed target peptide, polypeptide, or protein.
3. The method of claim 1 , where said adsorption-based, non-affinity chromatography column is an ion exchange chromatography column.
4. The method of claim 1 , wherein said recombinantly expressed target peptide, polypeptide, or protein is an endogenous peptide, polypeptide, or protein of said E. coli , or a heterologous peptide, polypeptide, or protein.
5. The method of claim 4 , wherein:
said recombinantly expressed endogenous peptide, polypeptide, or protein is selected from the group consisting of a nuclease, a ligase, a polymerase, an RNA- or DNA-modifying enzyme, a carbohydrate-modifying enzyme, an isomerase, a proteolytic enzyme, and a lipolytic enzyme, and
said recombinantly expressed heterologous peptide, polypeptide, or protein is selected from the group consisting of an enzyme, a therapeutic peptide, a therapeutic polypeptide, and a therapeutic protein.
6. The method of claim 5 , wherein:
said recombinantly expressed heterologous enzyme is selected from the group consisting of a nuclease, a ligase, a polymerase, an RNA- or DNA-modifying enzyme, a carbohydrate-modifying enzyme, an isomerase, a proteolytic enzyme, and a lipolytic enzyme, and
said recombinantly expressed therapeutic peptide, polypeptide, or protein is selected from the group consisting of antibody, an antibody fragment, a vaccine, an enzyme, a growth factor, a blood clotting factor, a hormone, a nerve factor, an interferon, an interleukin, lung surfactant protein, serum albumin, tissue plasminogen activator, and tumor necrosis factor.
7. The method of claim 6 , wherein said recombinantly expressed therapeutic enzyme is selected from the group consisting of α 1 -Antitrypsin, deoxyribonuclease, and superoxide dismutase.
8. The method of claim 1 , wherein said non-recombinantly expressed peptide, polypeptide, or protein is an endogenous peptide, polypeptide, or protein of said E. coli.
9. The method of claim 8 , wherein said non-recombinantly expressed endogenous peptide, polypeptide, or protein is selected from the group consisting of a nuclease, a ligase, a polymerase, an RNA- or DNA-modifying enzyme, a carbohydrate-modifying enzyme, an isomerase, a proteolytic enzyme, and a lipolytic enzyme.
10. A method of enriching the amount of a target peptide, polypeptide, or protein relative to other peptides, polypeptides, or proteins present in an initial protein mixture comprising said target peptide, polypeptide, or protein, comprising the steps of:
1) recombinantly or non-recombinantly expressing said target peptide, polypeptide, or protein in E. coli wherein a combination of genes selected from the group consisting of the following combinations of genes is deleted:
ΔhldDΔusgΔrraA;
ΔhldDΔusgΔrraAΔcutA;
ΔhldDΔusgΔrraAΔcutAΔnagD;
ΔhldDΔusgΔrraAΔcutAΔnagDΔspeA;
ΔhldDΔusgΔrraAΔcutAΔnagDΔspeAΔgldA;
ΔhldDΔusgΔrraAΔcutAΔnagDΔspeAΔgldAΔglnA;
ΔhldDΔusgΔrraAΔcutAΔnagDΔspeAΔgldAΔglnAΔmetE;
ΔhldDΔusgΔrraAΔcutAΔnagDΔspeAΔgldAΔglnAΔmetEΔtgt;
ΔhldDΔusgΔrraAΔcutAΔnagDΔspeAΔgldAΔglnAΔmetEΔtgtΔargG;
ΔhldDΔusgΔrraAΔcutAΔnagDΔspeAΔgldAΔglnAΔmetEΔtgtΔargGΔtypA;
ΔhldDΔusgΔrraAΔcutAΔnagDΔspeAΔgldAΔglnAΔmetEΔtgtΔargGΔtypAΔentF;
ΔhldDΔusgΔrraAΔcutAΔnagDΔspeAΔgldAΔglnAΔmetEΔtgtΔargGΔtypAΔentFΔycaO;
ΔhldDΔusgΔrraAΔcutAΔnagDΔspeAΔgldAΔglnAΔmetEΔtgtΔargGΔtypAΔentFΔycaOΔslyD;
ΔhldDΔusgΔrraAΔcutAΔnagDΔspeAΔgldAΔglnAΔmetEΔtgtΔargGΔtypAΔentFΔycaOΔslyDΔgat Z;
ΔhldDΔusgΔrraAΔcutAΔnagDΔspeAΔgldAΔglnAΔmetEΔtgtΔargGΔtypAΔentFΔycaOΔslyDΔgat ZΔilvB;
ΔhldDΔusgΔrraAΔcutAΔnagDΔspeAΔgldAΔglnAΔmetEΔtgtΔargGΔtypAΔentFΔycaOΔslyDΔgat ZΔilvBΔglgP;
ΔhldDΔusgΔrraAΔcutAΔnagDΔspeAΔgldAΔglnAΔmetEΔtgtΔargGΔtypAΔentFΔycaOΔslyDΔgat ZΔilvBΔglgPΔnusA; and
ΔhldDΔusgΔrraAΔcutAΔnagDΔspeAΔgldAΔglnAΔmetEΔtgtΔargGΔtypAΔentFΔycaOΔslyDΔgat ZΔilvBΔglgPΔnusAΔmetH;
2) in the case where said expressed target peptide, polypeptide, or protein is not secreted from said E. coli , preparing a lysate of said E. coli containing said expressed target peptide, polypeptide, or protein, producing an initial expressed target peptide-, polypeptide-, or protein-containing mixture; or
3) in the case where said expressed target peptide, polypeptide, or protein is secreted from said E. coli , harvesting culture medium in which said E. coli is grown, containing said expressed target peptide, polypeptide, or protein, thereby obtaining an initial expressed target peptide-, polypeptide-, or protein-containing mixture;
4) chromatographing said initial expressed target peptide-, polypeptide-, or protein-containing mixture of step 2) or step 3) using an affinity chromatography column comprising an affinity ligand bound to a solid phase, or an adsorption-based, non-affinity chromatography medium-containing column, and collecting elution fractions, thereby obtaining one or more fractions containing an enriched amount of said expressed target peptide, polypeptide, or protein relative to other peptides, polypeptides, or proteins in said fraction compared to the amount of said expressed target peptide, polypeptide, or protein relative to other peptides, polypeptides, or proteins in said initial expressed target peptide-, polypeptide-, or protein-containing mixture.
11. The method of claim 10 , further comprising chromatographing an enriched fraction of step 4) to obtain said expressed target peptide, polypeptide, or protein in a desired degree of purity.
12. The method of claim 11 , further comprising recovering said expressed target peptide, polypeptide, or protein.
13. The method of claim 10 , wherein said adsorption-based, non-affinity chromatography column is an ion exchange chromatography column.
14. The method of claim 10 , wherein said recombinantly expressed target peptide, polypeptide, or protein is an endogenous peptide, polypeptide, or protein of said E. coli , or a heterologous peptide, polypeptide, or protein.
15. The method of claim 14 , wherein:
said recombinantly expressed endogenous peptide, polypeptide, or protein is selected from the group consisting of a nuclease, a ligase, a polymerase, an RNA- or DNA-modifying enzyme, a carbohydrate-modifying enzyme, an isomerase, a proteolytic enzyme, and a lipolytic enzyme, and
said recombinantly expressed heterologous peptide, polypeptide, or protein is selected from the group consisting of an enzyme, a therapeutic peptide, a therapeutic polypeptide, and a therapeutic protein.
16. The method of claim 15 , wherein:
said recombinantly expressed heterologous enzyme is selected from the group consisting of a nuclease, a ligase, a polymerase, an RNA- or DNA-modifying enzyme, a carbohydrate-modifying enzyme, an isomerase, a proteolytic enzyme, and a lipolytic enzyme, and
said recombinantly expressed therapeutic peptide, polypeptide, or protein is selected from the group consisting of antibody, an antibody fragment, a vaccine, an enzyme, a growth factor, a blood clotting factor, a hormone, a nerve factor, an interferon, an interleukin, lung surfactant protein, serum albumin, tissue plasminogen activator, and tumor necrosis factor.
17. The method of claim 16 , wherein said recombinantly expressed therapeutic enzyme is selected from the group consisting of α 1 -Antitrypsin, deoxyribonuclease, and superoxide dismutase.
18. The method of claim 10 , wherein said non-recombinantly expressed peptide, polypeptide, or protein is an endogenous peptide, polypeptide, or protein of said E. coli.
19. The method of claim 18 , wherein said non-recombinantly expressed endogenous peptide, polypeptide, or protein is selected from the group consisting of a nuclease, a ligase, a polymerase, an RNA- or DNA-modifying enzyme, a carbohydrate-modifying enzyme, an isomerase, a proteolytic enzyme, and a lipolytic enzyme.