E. coli separatome-based protein expression and purification platform

Patent metadata

Field	Value
Publication number	US-2016230177-A1
Application number	US-201415022852-A
Country	US
Kind code	A1
Filing date	Sep 17, 2014
Priority date	Sep 17, 2013
Publication date	Aug 11, 2016
Grant date	—

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

1 . An E. coli host cell for expression of a target host cell or target recombinant peptide, polypeptide, or protein,
wherein the chromatographic separation efficiency of said target host cell or target recombinant peptide, polypeptide, or protein expressed in said E. coli host cell is improved in an amount in the range of from about 5% to about 50%,
said E. coli host cell comprising:
a) a reduced genome,
b) modified genome, or
c) a genome in which expression of genes is reduced or completely inhibited,
wherein genes that are deleted, modified, or the expression of which is reduced or completely inhibited in said host cell code for peptides, polypeptides, or proteins that impair the chromatographic separation efficiency of said target host cell or target recombinant peptide, polypeptide, or protein expressed in said host cell, wherein said genes are selected from the group consisting of: the genes listed in Table 9, and combinations thereof; combinations of any of the genes listed in Tables 8 and 9 taken together; the genes listed in Table 14, and combinations thereof; and combinations of any of the genes listed in Tables 8, 9, and 14 taken together, wherein deletion, modification, or reduction or complete inhibition of expression of said genes improves the chromatographic separation efficiency of said target host cell or target recombinant peptide, polypeptide, or protein in an amount in the range of from about 5% to about 50% compared to chromatographic separation efficiency of said target host cell or target recombinant peptide, polypeptide, or protein in the presence of peptides, polypeptides, or proteins coded for by said genes that are deleted, modified, and/or the expression of which is reduced or completely inhibited in said E. coli host cell upon affinity or adsorption, non-affinity column chromatography of said target host cell or target recombinant peptide polypeptide, or protein.

2 . The E. coli host cell of claim 1 , which is a strain selected from the group consisting of strain K-12, strain B, strain C, and strain W.

3 . The E. coli host cell of claim 2 , wherein:
said E. coli strain K-12 is selected from the group consisting of W3110, DH10B, DH5alpha, DH1, MG1655, and BW2952; and said E. coli strain B is selected from the group consisting of B REL606, BL21, and BL21-DE3.

4 . The E. coli host cell of claim 1 , which is selected from the group consisting of:
Alpha-Select Bacteriophage T1-Resistant Gold Efficiency (F− deoR endA1 recA1 relA1 gyrA96 hsdR17(rk − , mk + ) supE44 thi-1 phoA Δ(lacZYA-argF)U169 Φ80lacZΔM15λ−), Alpha-Select Bacteriophage T1-Resistant Silver Efficiency (F− deoR endA1 recA1 relA1 gyrA96 hsdR17(rk − , mk + ) supE44 thi-1 phoA Δ(lacZYA-argF)U169 Φ80lacZΔM15λ−), Alpha-Select Bronze Efficiency (F− deoR endA1 recA1 relA1 gyrA96 hsdR17(rk−, mk+) supE44 thi-1 phoA Δ(lacZYA-argF)U169 Φ80lacZΔM15λ−), Alpha-Select (F− deoR endA1 recA1 relA1 gyrA96 hsdR17(rk−, mk+) supE44 thi-1 phoA Δ(lacZYA-argF)U169 Φ80lacZΔM15λ−), AG1 (endA1 recA1 gyrA96 thi-1 relA1 glnV44 hsdR17(r K − m K + )), AB1157 (thr-1, araC14, leuB6(Am), Δ(gpt-proA)62, lacY1, tsx-33, qsr′-0, glnV44(AS), galK2(Oc), LAM−, Rac-0, hisG4(Oc), rfbC1, mgl-51, rpoS396(Am), rpsL31(strR), kdgK51, xylA5, mtl-1, argE3(Oc), thi-1), B2155 (thrB1004 pro thi strA hsdsS lacZD M15 (F′lacZD M15 lacI q traD36 proA + proB + ) Δ dapA::erm (Erm r ) pir::RP4 [::kan (Km r ) from SM10]), B834(DE3) (F − ompT hsdS B (r B − m B − ) gal dcm met (DE3)), BIOBlue (recA1 endA1 gyrA96 thi-1 hsdR17(rk−, mk+) supE44 relA1 lac [F′ proAB lacI q ZΔM15 Tn10(Tet r )]), BL21 ( E. coli B F− dcm ompT hsdS(r B − m B −) gal [malB + ] K-12 (λ S )), BL21(AI) (F − ompT gal dcm lon hsdS B (r B − m B − ) araB::T7RNAP-tetA), BL21(DE3) (F − ompT gal dcm lon hsdS B (r B − m B − ) λ(DE3 [lacI lacUV5-T7 gene 1 ind1 sam7 nin5])), BL21 (DE3) pLysS (F− ompT hsdSB(rB−, mB−) gal dcm (DE3) pLysS (CamR)), BL21-T1R (F− ompT hsdSB(rB− mB−) gal dcm tonA), BNN93 (F − tonA21 thi-1 thr-1 leuB6 lacY1 glnV44 rfbC1 fhuA1 mcrB e14-(mcrA − ) hsdR(r K − m K + ) λ − ), BNN97 (BNN93 (λgt11)), BW26434 (Δ(araD-araB)567, Δ(lacA-lacZ)514(::kan), lacI p -4000(lacI q ), λ − , rpoS396(Am)?, rph-1, Δ(rhaD-rhaB)568, bsdR514), C600 (F − tonA21 thi-1 thr-1 leuB6 lacY1 glnV44 rfbC1 fhuA1λ − ), CAG597 (F − lacZ(am) pho(am) lyrT[supC(ts)] trp(am) rpsL(Str R ) rpoH(am)165 zhg::Tn10 mal(am)), CAG626 (F − lacZ(am) pho(am) lon trp(am) tyrT[supC(ts)] rpsL(Str R ) mal(am)), CAG629 (F − lacZ(am) pho(am) lon supC(ts) trp(am) rpsL rpoH(am)165 zhg::Tn10 mal(am)), CH3-Blue (F− ΔmcrA Δ(mrr-hsdRMS-mcrBC) Φ80lacZΔM15 ΔlacX74 recA1 endA1 ara Δ139 Δ(ara, leu)7697 galU galrpsL(Str R ) nupG λ−), CSH50 (F − λ − ara Δ(lac-pro) rpsL thi fimE::IS1), D1210 (HB101 lacI q lacY + ), dam-dcm-Bacteriophage T1-Resistant (F− dam-13:Tn9(Cam R )dcm-6 ara-14 hisG4 leuB6 thi-1 lacY1 galK2 galT22 glnV44 hsdR2 xylA5 mtl-1 rpsL136(Str R ) rtbD1 tonA31 tsx78 mcrA mcrB1), DB3.1 (F− gyrA462 endA1 glnV44 Δ(sr1-recA) mcrB mrr hsdS20(r B − , m B + ) ara14 galK2 lacY1 proA2 rpsL20(Sm r ) xy15 Δleu mtl1), DH1 (endA1 recA1 gyrA96 thi-1 glnV44 relA1 hsdR17(r K − m K + ) λ − ), DH5α Turbo (F′ proA+B+ lacI q Δ lacZ M15/fhuA2 Δ(lac-proAB) glnV gal R(zgb-210::Tn10)Tet S endA1 thi-1 Δ(hsdS-mcrB)5), DH12S (mcrA Δ(mrr-hsdRMS-mcrBC) φ80d lacZΔM15 ΔlacX74 recA1 deoR Δ(ara, leu)7697 araD139 galU galK rpsL F′ [proAB + lacI q ZΔM15]), DM1 (F− dam-13::Tn9(Cm R ) dcm− mcrB hsdR-M+ gal1 gal2 ara− lac− thr− leu− tonR tsxR Su0), E. CLONI® 5ALPHA (fhuA2Δ(argF-lacZ)U169 phoA glnV44 Φ80 Δ(lacZ)M15 gyrA96 recA1 relA1 endA1 thi-1 hsdR17), E. CLONI® 10G (F− mcrA Δ(mnr-hsdRMS-mcrBC) endA1 recA1 Φ80dlacZΔM15 ΔlacX74 araI139 Δ(ara,leu)7697galU galK rpsL nupG λ− tonA (StrR)), E. CLONI® 10GF′ ([F′ pro A+B+ lacI q ZΔM15::T10 (Tet R )]/mcrA Δ(mrr-hsdRMS-mcrBC) endA1 recA1 Φ80dlacZΔM15 ΔlacX74 araD139 Δ(ara, leu)7697 galU galK rpsL nupG λ− tonA (StrR)), E. coli K12 ER2738 (F′proA+B+ lacI q Δ(lacZ)M15 zzf::Tn10(Tet R )/fhuA2 glnV Δ(lac-proAB) thi-1 Δ(hsdS-mcrB)5), ElectroMax™ DH10B (F − mcrA Δ(mrr-hsdRMS-mcrBC) Φ80lacZΔM15 ΔlacX74 recA1 endA1 araD139Δ(ara,leu)7697 galU galK λ − rpsL nupG), ELECTROMAX™ DH5ALPHA-E (F− φ80lacZΔM15 Δ(lacZY A-argF) U169 recA1 endA1 hsdR17 (rk−, mk+) galphoA supE44λ-thi-1 gyrA96 relA1), ElectroSHOX (F− mcrA Δ(mrr-hsdRMS-mcrBC) Φ80lacZΔM15 ΔlacX74 recA1 endA1 ara Δ139 Δ(ara, leu)7697 galU galKrpsL(Str R ) nupG λ − ), EP-MAX™10B F′ (mcrA Δ(mrr-hsdRMS-mcrBC) φ80dlacZΔM15 ΔlacX74 deoR recA1 endA1 araD139 Δ(ara, leu)7697 galU galK rpsL nupG λ−/F′[lacI q ZΔM15 Tn10 (Tet R )]), ER1793 (F − fhuA2 Δ(lacZ)r1 glnV44 e14 − (McrA − ) trp-31 his-1 rpsL104 xyl-7 mtl-2 metB1 Δ(mcrC-mrr)114::IS10), ER1821 (F − glnV44 e14 − (McrA − ) rfbD1? rel4? endA1 spoT1? thi-1 Δ(mcrC-mrr))114::IS10), ER2738 (F′proA + B + lacI q Δ(lacZ)M15 zzf::Tn10(Tet R )/fhuA2 glnV Δ(lac-proAB) thi-1 Δ(hsdS-mcrB)5), ER2267 (F′ proA + B + lacI q Δ(lacZ)M15 zzf::mini-Tn10 (Kan R )/Δ(argF-lacZ)U169 glnV44 e14 − (McrA − ) rfbD1? recA1 relA1? endA1 spoT1! thi-1 Δ(mcrC-mrr)114::IS10), ER2507 (F − ara-14 leuB6 fhuA2 Δ(argF-lac)U169 lacY1 glnV44 galK2 rpsL20 xyl-5 mtl-5 Δ(malB) zjc::Tn5(Kan R )Δ(mcrC-mrr) HB101 ), ER2508 (F − ara-14 leuB6 fhuA2 Δ(argF-lac)U169 lacY1 lon::miniTn10(Tet R ) glnV44 galK2 rpsL20(Str R ) xyl-5 mtl-5 Δ(malB) zjc::Tn5(Kan R ) Δ(mcrC-mrr) HB101 ), ER2738 (F′proA + B + lacI q Δ(lacZ)M15 zzf::Tn10(Tet R )/fhuA2 glnV Δ(lac-proAB) thi-1 Δ(hsdS-mcrB)5), ER2925 (ara-14 leuB6 fhuA31 lacY1 tsx78 glnV44 galK2 galT22 mcrA dcm-6 hisG4 rfbD1 R(zgb210::Tn10)Tet S endA1 rpsL136 dam13::Tn9 xylA-5 mtl-1 thi-1 mcrB1 hsdR2), GC5™ (:F− Φ80lacZ Δ M15 Δ (lacZYA-argF)U169 endA1 recA1 relA1 gyrA96 hsdR17 (r k − , m k + ) phoA supE44 thi-1λ−T1R), GC10 (F− mcrA Δ(mrr-hsdRMSmcrBC)

Assignees

• Brune Ellen M
• Beitle Robert R
• Atall Mohammad M
• Bartlow Patrick R
• Henry Ralph L
• Univ Arkansas

Inventors

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

Classifications

• C12N15/70 Primary

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

• C12P21/00

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

• C12P21/02

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