High-level accumulation of a recombinant antibody fragment in the periplasm of Escherichia coli requires a triple-mutant (degP prc spr) host strain

被引:63
作者
Chen, C
Snedecor, B
Nishihara, JC
Joly, JC
McFarland, N
Andersen, DC
Battersby, JE
Champion, KM [1 ]
机构
[1] Genentech Inc, Dept Analyt Chem, San Francisco, CA 94080 USA
[2] Genentech Inc, Dept Cell Culture & Fermentat Res & Dev, San Francisco, CA 94080 USA
关键词
Escherichia coli; periplasmic protease; recombinant antibody; biopharmaceutical; two-dimensional gel electrophoresis; high cell density fermentation;
D O I
10.1002/bit.20014
中图分类号
Q81 [生物工程学(生物技术)]; Q93 [微生物学];
学科分类号
071005 ; 0836 ; 090102 ; 100705 ;
摘要
During production of a humanized antibody fragment secreted into the periplasm of Escherichia coli, proteolytic degradation of the light chain was observed. In order to determine which protease(s) were responsible for this degradation, we compared expression of the F(ab')(2) antibody fragment in several E. coli strains carrying mutations in genes encoding periplasmic proteases. Analysis of strains cultured in high cell density fermentations showed that the combination of mutations in degP prc spr was necessary for the cells to produce high levels of the desired recombinant antibody fragment. In order to eliminate the possible effects of mutations in other genes, we constructed E. coli strains with protease mutations in isogenic backgrounds and repeated the studies in high cell density fermentations. Extensive light chain proteolysis persisted in degP strains. However, light chain proteolysis was substantially decreased in jorc and jorc spr strains, and was further decreased with the introduction of a degP mutation in prc and prc spr mutant strains. These results show that the periplasmic protease Prc (Tsp) is primarily responsible for proteolytic degradation of the light chain during expression of a recombinant antibody fragment in E. coli, and that DegP (HtrA) makes a minor contribution to this degradation as well. The results also show that spr, a suppressor of growth defects in prc strains, is required for a prc mutant to survive throughout high cell density fermentations. (C) 2004 Wiley Periodicals, Inc.
引用
收藏
页码:463 / 474
页数:12
相关论文
共 31 条
  • [1] Pharmacokinetics of rhuMAb CD18, a recombinant humanised monoclonal antibody fragment to CD18, in normal healthy human volunteers
    Allison, DE
    Gourlay, SG
    Koren, E
    Miller, RM
    Fox, JA
    [J]. BIODRUGS, 2002, 16 (01) : 63 - 70
  • [2] DEGRADATION OF SECRETED PROTEINS IN ESCHERICHIA-COLI
    BANEYX, F
    GEORGIOU, G
    [J]. ANNALS OF THE NEW YORK ACADEMY OF SCIENCES-SERIES, 1992, 665 : 301 - 308
  • [3] CONSTRUCTION AND CHARACTERIZATION OF ESCHERICHIA-COLI STRAINS DEFICIENT IN MULTIPLE SECRETED PROTEASES - PROTEASE-III DEGRADES HIGH-MOLECULAR-WEIGHT SUBSTRATES INVIVO
    BANEYX, F
    GEORGIOU, G
    [J]. JOURNAL OF BACTERIOLOGY, 1991, 173 (08) : 2696 - 2703
  • [4] Affinity-reversed-phase liquid chromatography assay to quantitate recombinant antibodies and antibody fragments in fermentation broth
    Battersby, JE
    Snedecor, B
    Chen, C
    Champion, KM
    Riddle, L
    Vanderlaan, M
    [J]. JOURNAL OF CHROMATOGRAPHY A, 2001, 927 (1-2) : 61 - 76
  • [5] HUMANIZATION OF AN ANTI-P185HER2 ANTIBODY FOR HUMAN CANCER-THERAPY
    CARTER, P
    PRESTA, L
    GORMAN, CM
    RIDGWAY, JBB
    HENNER, D
    WONG, WLT
    ROWLAND, AM
    KOTTS, C
    CARVER, ME
    SHEPARD, HM
    [J]. PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 1992, 89 (10) : 4285 - 4289
  • [6] THE ACYLATED PRECURSOR FORM OF THE COLICIN-A LYSIS PROTEIN IS A NATURAL SUBSTRATE OF THE DEGP PROTEASE
    CAVARD, D
    LAZDUNSKI, C
    HOWARD, SP
    [J]. JOURNAL OF BACTERIOLOGY, 1989, 171 (11) : 6316 - 6322
  • [7] Comparison of the Escherichia coli proteomes for recombinant human growth hormone producing and nonproducing fermentations
    Champion, KM
    Nishihara, JC
    Aldor, IS
    Moreno, GT
    Andersen, D
    Stults, KL
    Vanderlaan, M
    [J]. PROTEOMICS, 2003, 3 (07) : 1365 - 1373
  • [8] Champion KM, 2001, PROTEOMICS, V1, P1133, DOI 10.1002/1615-9861(200109)1:9<1133::AID-PROT1133>3.0.CO
  • [9] 2-S
  • [10] COVELL DG, 1986, CANCER RES, V46, P3969