CoChat
Sign Up
Cited by 49Heat shock proteins researchProtein purification and stabilityBacterial Genetics and Biotechnology

The DOI matches its Crossref record.

Co-expression of Skp and FkpA chaperones improves cell viability and alters the global expression of stress response genes during scFvD1.3 production

Open access
Ow, Dave, Lim, Denis, Morin Nissom, Peter, Camattari, Andrea, Wong, Victor·2010·Microbial Cell Factories
DOI 10.1186/1475-2859-9-22PMC PMC2868799
Open this paper in CoChat — free accountOpen PDF

Abstract

BACKGROUND: The overexpression of scFv antibody fragments in the periplasmic space of Escherichia coli frequently results in extensive protein misfolding and loss of cell viability. Although protein folding factors such as Skp and FkpA are often exploited to restore the solubility and functionality of recombinant protein products, their exact impact on cellular metabolism during periplasmic antibody fragment expression is not clearly understood. In this study, we expressed the scFvD1.3 antibody fragment in E. coli BL21 and evaluated the overall physiological and global gene expression changes upon Skp or FkpA co-expression. RESULTS: The periplasmic expression of scFvD1.3 led to a rapid accumulation of insoluble scFvD1.3 proteins and a decrease in cell viability. The co-expression of Skp and FkpA improved scFvD1.3 solubility and cell viability in a dosage-dependent manner. Through mutagenesis experiments, it was found that only the chaperone activity of FkpA, not the peptidyl-prolyl isomerase (PPIase) activity, is required for the improvement in cell viability. Global gene expression analysis of the scFvD1.3 cells over the chaperone-expressing cells showed a clear up-regulation of genes involved in heat-shock and misfolded protein stress responses. These included genes of the major HSP70 DnaK chaperone family and key proteases belonging to the Clp and Lon protease systems. Other metabolic gene expression trends include: (1) the differential regulation of several energy metabolic genes, (2) down-regulation of the central metabolic TCA cycle and transport genes, and (3) up-regulation of ribosomal genes. CONCLUSIONS: The simultaneous activation of multiple stress related and other metabolic genes may constitute the stress response to protein misfolding in the scFvD1.3 cells. These gene expression information could prove to be valuable for the selection and construction of reporter contructs to monitor the misfolded protein stress response during antibody fragment production.

Overview

The claims and statistics of this paper haven’t been checked yet.

Run the check — free, no account needed. It reads the open-access text and checks the sources it cites.

A few free checks a day · papers already verified open instantly · results are public

Integrity signals

Registry

Crossref · 10.1186/1475-2859-9-22

Cites

Statistics

Verification

Claims the paper makes

The claims and statistics of this paper haven’t been checked yet.

Run the check — free, no account needed. It reads the open-access text and checks the sources it cites.

A few free checks a day · papers already verified open instantly · results are public

Claims other papers make about this one

Each is checked against this paper’s own text: does it say what the citing paper says it does?

  • Coexpression of an scFv with the periplasmic chaperones FkpA and Skp significantly improved the cytoplasmic solubility of the scFv and cell viability.

    “In another study, the coexpression of an scFv with the periplasmic chaperones FkpA and Skp significantly improved the cytoplasmic solubility of the scFv and cell viability [168,172].”

    From Evolution of Escherichia coli Expression System in Producing Antibody Recombinant Fragments (2020) · cites this paper as [168]

    The Conclusions and Results and discussion report increased scFvD1.3 solubility and cell viability with Skp or FkpA co-expression. They describe periplasmic scFv expression, however, not improved cytoplasmic solubility. Page numbers were not provided in our copy. (LLM, conf=0.90)

    Experimental — accuracy not yet measured

Self-check

Percentages, sums and reported differences re-computed from the paper’s own numbers.

The self-check needs the paper’s full text.

Citations

Cites

No references could be resolved yet.

Research works printed but not linked: 72

  1. Holliger P Hudson PJ Engineered antibody fragments and the rise of single domains Nat Biotechnol 2005 23 1126 1136 10.1038/nbt1142 16151406 · DOI
  2. Hudson PJ Recombinant antibody constructs in cancer therapy Curr Opin Immunol 1999 11 548 557 10.1016/S0952-7915(99)00013-8 10508712 · DOI
  3. Viti F Tarli L Giovannoni L Zardi L Neri D Increased binding affinity and valence of recombinant antibody fragments lead to improved targeting of tumoral angiogenesis Cancer Res 1999 59 347 352 9927045
  4. Campana V Zentilin L Mirabile I Kranjc A Casanova P Giacca M Development of antibody fragments for immunotherapy of prion diseases Biochem J 2009 418 507 515 10.1042/BJ20081541 19000036 · DOI
  5. Arbabi-Ghahroudi M Tanha J MacKenzie R Prokaryotic expression of antibodies Cancer Metastasis Rev 2005 24 501 519 10.1007/s10555-005-6193-1 16408159 · DOI
  6. Gasser B Saloheimo M Rinas U Dragosits M Rodriguez-Carmona E Baumann K Protein folding and conformational stress in microbial cells producing recombinant proteins: a host comparative overview Microb Cell Fact 2008 7 11 10.1186/1475-2859-7-11 18394160 PMC2322954 · DOI
  7. Baneyx F Mujacic M Recombinant protein folding and misfolding in Escherichia coli Nat Biotechnol 2004 22 1399 1408 10.1038/nbt1029 15529165 · DOI
  8. Kolaj O Spada S Robin S Wall JG Use of folding modulators to improve heterologous protein production in Escherichia coli Microb Cell Fact 2009 8 9 10.1186/1475-2859-8-9 19173718 PMC2642769 · DOI
  9. Walton TA Sousa MC Crystal structure of Skp, a prefoldin-like chaperone that protects soluble and membrane proteins from aggregation Mol Cell 2004 15 367 374 10.1016/j.molcel.2004.07.023 15304217 · DOI
  10. Bulieris PV Behrens S Holst O Kleinschmidt JH Folding and insertion of the outer membrane protein OmpA is assisted by the chaperone Skp and by lipopolysaccharide J Biol Chem 2003 278 9092 9099 10.1074/jbc.M211177200 12509434 · DOI
  11. Hayhurst A Harris WJ Escherichia coli skp chaperone coexpression improves solubility and phage display of single-chain antibody fragments Protein Expr Purif 1999 15 336 343 10.1006/prep.1999.1035 10092493 · DOI
  12. Horne SM Young KD Escherichia coli and other species of the Enterobacteriaceae encode a protein similar to the family of Mip-like FK506-binding proteins Arch Microbiol 1995 163 357 365 10.1007/BF00404209 7540828 · DOI
  13. Saul FA Arie JP Vulliez-le Normand B Kahn R Betton JM Bentley GA Structural and functional studies of FkpA from Escherichia coli, a cis/trans peptidyl-prolyl isomerase with chaperone activity J Mol Biol 2004 335 595 608 10.1016/j.jmb.2003.10.056 14672666 · DOI · Paper page
  14. Missiakas D Betton JM Raina S New components of protein folding in extracytoplasmic compartments of Escherichia coli SurA, FkpA and Skp/OmpH Mol Microbiol 1996 21 871 884 10.1046/j.1365-2958.1996.561412.x 8878048 · DOI
  15. Arie JP Sassoon N Betton JM Chaperone function of FkpA, a heat shock prolyl isomerase, in the periplasm of Escherichia coli Mol Microbiol 2001 39 199 210 10.1046/j.1365-2958.2001.02250.x 11123702 · DOI
  16. Zhang Z Song LP Fang M Wang F He D Zhao R Production of soluble and functional engineered antibodies in Escherichia coli improved by FkpA Biotechniques 2003 35 1032 14628677
  17. Boulot G Eisele JL Bentley GA Bhat TN Ward ES Winter G Crystallization and preliminary X-ray diffraction study of the bacterially expressed Fv from the monoclonal anti-lysozyme antibody D1.3 and of its complex with the antigen, lysozyme J Mol Biol 1990 213 617 619 10.1016/S0022-2836(05)80248-7 2113587 · DOI
  18. Hawkins RE Russell SJ Baier M Winter G The contribution of contact and non-contact residues of antibody in the affinity of binding to antigen. The interaction of mutant D1.3 antibodies with lysozyme J Mol Biol 1993 234 958 964 10.1006/jmbi.1993.1650 8263942 · DOI
  19. England P Bregegere F Bedouelle H Energetic and kinetic contributions of contact residues of antibody D1.3 in the interaction with lysozyme Biochemistry 1997 36 164 172 10.1021/bi961419y 8993330 · DOI
  20. Aldor IS Krawitz DC Forrest W Chen C Nishihara JC Joly JC Proteomic profiling of recombinant Escherichia coli in high-cell-density fermentations for improved production of an antibody fragment biopharmaceutical Appl Environ Microbiol 2005 71 1717 1728 10.1128/AEM.71.4.1717-1728.2005 15811994 PMC1082529 · DOI
  21. Bardwell JC McGovern K Beckwith J Identification of a protein required for disulfide bond formation in vivo Cell 1991 67 581 589 10.1016/0092-8674(91)90532-4 1934062 · DOI
  22. Fabianek RA Hennecke H Thony-Meyer L Periplasmic protein thiol:disulfide oxidoreductases of Escherichia coli FEMS Microbiol Rev 2000 24 303 316 10.1111/j.1574-6976.2000.tb00544.x 10841975 · DOI
  23. Ramm K Pluckthun A The periplasmic Escherichia coli peptidylprolyl cis, trans-isomerase FkpA. II. Isomerase-independent chaperone activity in vitro J Biol Chem 2000 275 17106 17113 10.1074/jbc.M910234199 10748201 · DOI
  24. Bothmann H Pluckthun A The periplasmic Escherichia coli peptidylprolyl cis, trans-isomerase FkpA. I. Increased functional expression of antibody fragments with and without cis-prolines J Biol Chem 2000 275 17100 17105 10.1074/jbc.M910233199 10748200 · DOI · Paper page
  25. Kanemori M Mori H Yura T Induction of heat shock proteins by abnormal proteins results from stabilization and not increased synthesis of sigma 32 in Escherichia coli J Bacteriol 1994 176 5648 5653 7916010 PMC196767
  26. Wild J Walter WA Gross CA Altman E Accumulation of secretory protein precursors in Escherichia coli induces the heat shock response J Bacteriol 1993 175 3992 3997 8320215 PMC204827
  27. Gross CA Neidhardt FC, Curtiss R, Lin ECC, Low KB, Magasanik B, Reanikoff WS, et al Function and regulation of the heat shock Escherichia coli and Salmonella 1996 ASM Press (Washinton DC) 1382 1399
  28. Bukau B Horwich AL The Hsp70 and Hsp60 chaperone machines Cell 1998 92 351 366 10.1016/S0092-8674(00)80928-9 9476895 · DOI
  29. Genevaux P Georgopoulos C Kelley WL The Hsp70 chaperone machines of Escherichia coli: a paradigm for the repartition of chaperone functions Mol Microbiol 2007 66 840 857 10.1111/j.1365-2958.2007.05961.x 17919282 · DOI
  30. Laskowska E Wawrzynow A Taylor A IbpA and IbpB, the new heat-shock proteins, bind to endogenous Escherichia coli proteins aggregated intracellularly by heat shock Biochimie 1996 78 117 122 10.1016/0300-9084(96)82643-5 8818220 · DOI
  31. Han MJ Yun H Lee SY Microbial small heat shock proteins and their use in biotechnology Biotechnol Adv 2008 26 591 609 10.1016/j.biotechadv.2008.08.004 18789382 · DOI
  32. Maurizi MR Proteases and protein degradation in Escherichia coli Experientia 1992 48 178 201 10.1007/BF01923511 1740190 · DOI
  33. Goldberg AL Moerschell RP Chung CH Maurizi MR ATP-dependent protease La (lon) from Escherichia coli Methods Enzymol 1994 244 350 375 full_text 7845219
  34. Wang J Hartling JA Flanagan JM The structure of ClpP at 2.3 A resolution suggests a model for ATP-dependent proteolysis Cell 1997 91 447 456 10.1016/S0092-8674(00)80431-6 9390554 · DOI
  35. Dougan DA Reid BG Horwich AL Bukau B ClpS, a substrate modulator of the ClpAP machine Mol Cell 2002 9 673 683 10.1016/S1097-2765(02)00485-9 11931773 · DOI
  36. Chung CH Goldberg AL The product of the lon (capR) gene in Escherichia coli is the ATP-dependent protease, protease La Proc Natl Acad Sci USA 1981 78 4931 4935 10.1073/pnas.78.8.4931 6458037 PMC320299 · DOI
  37. Laskowska E Kuczynska-Wisnik D Skorko-Glonek J Taylor A Degradation by proteases Lon, Clp and HtrA, of Escherichia coli proteins aggregated in vivo by heat shock; HtrA protease action in vivo and in vitro Mol Microbiol 1996 22 555 571 10.1046/j.1365-2958.1996.1231493.x 8939438 · DOI
  38. Ryzhavskaia AS Kaidalova NV Anukhin I Kliachko EV Livshits VA [Stability of normal, abnormal and recombinant proteins in Escherichia coli strains deficient for intracellular proteinase La--the product of the lon gene] Mol Biol (Mosk) 1988 22 201 208 3287135
  39. Wu WF Zhou Y Gottesman S Redundant in vivo proteolytic activities of Escherichia coli Lon and the ClpYQ (HslUV) protease J Bacteriol 1999 181 3681 3687 10368141 PMC93844
  40. Rohrwild M Coux O Huang HC Moerschell RP Yoo SJ Seol JH HslV-HslU: A novel ATP-dependent protease complex in Escherichia coli related to the eukaryotic proteasome Proc Natl Acad Sci USA 1996 93 5808 5813 10.1073/pnas.93.12.5808 8650174 PMC39143 · DOI
  41. Kornitzer D Teff D Altuvia S Oppenheim AB Isolation, characterization, and sequence of an Escherichia coli heat shock gene, htpX J Bacteriol 1991 173 2944 2953 1826904 PMC207877
  42. Shimohata N Chiba S Saikawa N Ito K Akiyama Y The Cpx stress response system of Escherichia coli senses plasma membrane proteins and controls HtpX, a membrane protease with a cytosolic active site Genes Cells 2002 7 653 662 10.1046/j.1365-2443.2002.00554.x 12081643 · DOI
  43. Missiakas D Georgopoulos C Raina S The Escherichia coli dsbC (xprA) gene encodes a periplasmic protein involved in disulfide bond formation EMBO J 1994 13 2013 2020 8168498 PMC395044
  44. Dartigalongue C Missiakas D Raina S Characterization of the Escherichia coli sigma E regulon J Biol Chem 2001 276 20866 20875 10.1074/jbc.M100464200 11274153 · DOI
  45. Park JH Lee YS Chung CH Goldberg AL Purification and characterization of protease Re, a cytoplasmic endoprotease in Escherichia coli J Bacteriol 1988 170 921 926 2892828 PMC210743
  46. Keiler KC Silber KR Downard KM Papayannopoulos IA Biemann K Sauer RT C-terminal specific protein degradation: activity and substrate specificity of the Tsp protease Protein Sci 1995 4 1507 1515 10.1002/pro.5560040808 8520476 PMC2143181 · DOI
  47. Spiers A Lamb HK Cocklin S Wheeler KA Budworth J Dodds AL PDZ domains facilitate binding of high temperature requirement protease A (HtrA) and tail-specific protease (Tsp) to heterologous substrates through recognition of the small stable RNA A (ssrA)-encoded peptide J Biol Chem 2002 277 39443 39449 10.1074/jbc.M202790200 12177052 · DOI
  48. Hottenrott S Schumann T Pluckthun A Fischer G Rahfeld JU The Escherichia coli SlyD is a metal ion-regulated peptidyl-prolyl cis/trans-isomerase J Biol Chem 1997 272 15697 15701 10.1074/jbc.272.25.15697 9188461 · DOI
  49. Rahfeld JU Schierhorn A Mann K Fischer G A novel peptidyl-prolyl cis/trans isomerase from Escherichia coli FEBS Lett 1994 343 65 69 10.1016/0014-5793(94)80608-X 8163020 · DOI
  50. Korber P Zander T Herschlag D Bardwell JC A new heat shock protein that binds nucleic acids J Biol Chem 1999 274 249 256 10.1074/jbc.274.1.249 9867837 · DOI
  51. Korber P Stahl JM Nierhaus KH Bardwell JC Hsp15: a ribosome-associated heat shock protein EMBO J 2000 19 741 748 10.1093/emboj/19.4.741 10675343 PMC305612 · DOI
  52. Glick BR Metabolic load and heterologous gene expression Biotechnol Adv 1995 13 247 261 10.1016/0734-9750(95)00004-A 14537822 · DOI
  53. Somerville JE Jr Goshorn SC Fell HP Darveau RP Bacterial aspects associated with the expression of a single-chain antibody fragment in Escherichia coli Appl Microbiol Biotechnol 1994 42 595 603 10.1007/BF00173926 7765735 · DOI
  54. Calhoun MW Gennis RB Demonstration of separate genetic loci encoding distinct membrane-bound respiratory NADH dehydrogenases in Escherichia coli J Bacteriol 1993 175 3013 3019 8387992 PMC204620
  55. Puustinen A Finel M Haltia T Gennis RB Wikstrom M Properties of the two terminal oxidases of Escherichia coli Biochemistry 1991 30 3936 3942 10.1021/bi00230a019 1850294 · DOI
  56. Schneider D Pohl T Walter J Dorner K Kohlstadt M Berger A Assembly of the Escherichia coli NADH:ubiquinone oxidoreductase (complex I) Biochim Biophys Acta 2008 1777 735 739 10.1016/j.bbabio.2008.03.003 18394423 · DOI
  57. Iverson TM Luna-Chavez C Cecchini G Rees DC Structure of the Escherichia coli fumarate reductase respiratory complex Science 1999 284 1961 1966 10.1126/science.284.5422.1961 10373108 · DOI
  58. Cunningham L Gruer MJ Guest JR Transcriptional regulation of the aconitase genes (acnA and acnB) of Escherichia coli Microbiology 1997 143 Pt 12 3795 3805 10.1099/00221287-143-12-3795 9421904 · DOI
  59. Brown TD Jones-Mortimer MC Kornberg HL The enzymic interconversion of acetate and acetyl-coenzyme A in Escherichia coli J Gen Microbiol 1977 102 327 336 21941
  60. Ishihama A Functional modulation of Escherichia coli RNA polymerase Annu Rev Microbiol 2000 54 499 518 10.1146/annurev.micro.54.1.499 11018136 · DOI
  61. Lindahl L Zengel JM Ribosomal genes in Escherichia coli Annu Rev Genet 1986 20 297 326 10.1146/annurev.ge.20.120186.001501 2434021 · DOI
  62. Maguire BA Zimmermann RA The ribosome in focus Cell 2001 104 813 816 10.1016/S0092-8674(01)00278-1 11290319 · DOI
  63. Inokuchi H Yamao F Soll D, Rajbhandary UL, Rajbhandary TL Structure and expression of prokaryotic tRNA tRNA: Structure, Biosynthesis, and function 1995 ASM Press, Washington DC 17 30
  64. Charollais J Dreyfus M Iost I CsdA, a cold-shock RNA helicase from Escherichia coli, is involved in the biogenesis of 50S ribosomal subunit Nucleic Acids Res 2004 32 2751 2759 10.1093/nar/gkh603 15148362 PMC419605 · DOI
  65. Wintermeyer W Savelsbergh A Semenkov YP Katunin VI Rodnina MV Mechanism of elongation factor G function in tRNA translocation on the ribosome Cold Spring Harb Symp Quant Biol 2001 66 449 458 10.1101/sqb.2001.66.449 12762047 · DOI
  66. Goldstein JL Caskey CT Peptide chain termination: effect of protein S on ribosomal binding of release factors Proc Natl Acad Sci USA 1970 67 537 543 10.1073/pnas.67.2.537 5289007 PMC283241 · DOI
  67. Bylund GO Wipemo LC Lundberg LA Wikstrom PM RimM and RbfA are essential for efficient processing of 16S rRNA in Escherichia coli J Bacteriol 1998 180 73 82 9422595 PMC106851
  68. Liu X Matsumura P The FlhD/FlhC complex, a transcriptional activator of the Escherichia coli flagellar class II operons J Bacteriol 1994 176 7345 7351 7961507 PMC197124
  69. Ramseier TM Bledig S Michotey V Feghali R Saier MH Jr The global regulatory protein FruR modulates the direction of carbon flow in Escherichia coli Mol Microbiol 1995 16 1157 1169 10.1111/j.1365-2958.1995.tb02339.x 8577250 · DOI
  70. Ramseier TM Cra and the control of carbon flux via metabolic pathways Res Microbiol 1996 147 489 493 10.1016/0923-2508(96)84003-4 9084760 · DOI
  71. Ow DSW Nissom PM Philp R Oh SKW Yap MGS Global transcriptional analysis of metabolic burden due to plasmid maintenance in Escherichia coli DH5a during batch fermentation Enzyme Microb Technol 2006 39 391 398 10.1016/j.enzmictec.2005.11.048 · DOI
  72. Ow DSW Lee RMY Philp R Nissom PM Oh SKW Yap MGS Inactivating FruR global regulator in plasmid bearing Escherichia coli alters metabolic gene expression and improves growth rate J Biotechnol 2007 131 261 269 10.1016/j.jbiotec.2007.07.508 17719117 · DOI

Cited by

Sorted by citations

Metadata

Authors
Ow, D., Lim, D., Morin Nissom, P., Camattari, A., & Wong, V.
Affiliations
Agency for Science, Technology and Research (SG)
Corresponding author
Ow, Dave
ORCID
Venue
Microbial Cell Factories
Publisher
Springer Science and Business Media LLC
Published
2010
Volume
9
Issue
1
Pages
22
ISSN
1475-2859 (ISSN-L)
DOI
10.1186/1475-2859-9-22
PMC
PMC2868799
OpenAlex
W2110751397
Cited by
49
References
0
Citations by year
2026: 1 · 2025: 6 · 2024: 1 · 2023: 1 · 2022: 3 · 2021: 5 · 2020: 8 · 2019: 1 · 2017: 1 · 2016: 2 · 2015: 4 · 2014: 4 · 2013: 5 · 2012: 3
FWCI
1.56
Type
journal article
Language
en
Open access
Licence
cc-by
Locations
  • Microbial Cell Factories (publishedVersion, cc-by)
  • Europe PMC (PubMed Central) (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
Funders
Agency for Science, Technology and Research
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Heat shock proteins research
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Protein purification and stability
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Bacterial Genetics and Biotechnology
Keywords
Skp chaperone, single-chain antibody fragments, protein misfolding, cell viability, chaperone co-expression, heat shock response, Clp protease, Lon protease, proteasome, global gene expression, energy metabolism, TCA cycle, ribosomal genes, reporter constructs
MeSH terms
Cell Survival, Cloning, Molecular, DNA-Binding Proteins, Escherichia coli, Membrane Proteins, Stress, Physiological, Gene Expression Regulation, Bacterial, Protein Folding, Molecular Chaperones, Peptidylprolyl Isomerase, Escherichia coli Proteins, Single-Chain Antibodies

About these checks

Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.

Statistics and arithmetic are recomputed, not judged.

Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.

What counts as a claim ›How we measure confidence ›How we check this ›

Are you an author, or do you see a problem with this page? Request a review