CoChat
Sign Up
Cited by 118Protein purification and stabilityHeat shock proteins researchProtein Structure and Dynamics

Kinetics of Inclusion Body Formation and Its Correlation with the Characteristics of Protein Aggregates in Escherichia coli

Open access
Upadhyay, Arun K., Murmu, Aruna, Singh, Anupam, Panda, Amulya K.·2012·PLoS ONE
DOI 10.1371/journal.pone.0033951PMC PMC3315509
Open this paper in CoChat — free accountOpen PDF

Abstract

The objective of the research was to understand the structural determinants governing protein aggregation into inclusion bodies during expression of recombinant proteins in Escherichia coli. Recombinant human growth hormone (hGH) and asparaginase were expressed as inclusion bodies in E.coli and the kinetics of aggregate formation was analyzed in details. Asparaginase inclusion bodies were of smaller size (200 nm) and the size of the aggregates did not increase with induction time. In contrast, the seeding and growth behavior of hGH inclusion bodies were found to be sequential, kinetically stable and the aggregate size increased from 200 to 800 nm with induction time. Human growth hormone inclusion bodies showed higher resistance to denaturants and proteinase K degradation in comparison to those of asparaginase inclusion bodies. Asparaginase inclusion bodies were completely solubilized at 2-3 M urea concentration and could be refolded into active protein, whereas 7 M urea was required for complete solubilization of hGH inclusion bodies. Both hGH and asparaginase inclusion bodies showed binding with amyloid specific dyes. In spite of its low β-sheet content, binding with dyes was more prominent in case of hGH inclusion bodies than that of asparaginase. Arrangements of protein molecules present in the surface as well as in the core of inclusion bodies were similar. Hydrophobic interactions between partially folded amphiphillic and hydrophobic alpha-helices were found to be one of the main determinants of hGH inclusion body formation. Aggregation behavior of the protein molecules decides the nature and properties of inclusion bodies.

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 · 10.1371/journal.pone.0033951

Cites

Statistics

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?

  • High local concentrations of nascent heterologous protein together with insufficient amount of folding-promoting chaperons may lead to partially folded or misfolded protein intermediates that give rise to formation of insoluble protein aggregates known as inclusion bodies in both cytoplasm and periplasm.

    “High local concentrations of the nascent heterologous protein together with an insufficient amount of folding-promoting chaperons may lead to partially folded or misfolded protein intermediates that give rise to the formation, in both the cytoplasm and periplasm, of insoluble protein aggregates known as inclusion bodies, IBs [77].”

    From Evolution of Escherichia coli Expression System in Producing Antibody Recombinant Fragments (2020) · cites this paper as [77]

Self-check

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

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

Cites

No references could be resolved yet.

Research works printed but not linked: 43

  1. [1]Fahnert B Lilie H Neubauer P 2004 Inclusion bodies: formation and utilization. Adv Biochem Eng/Biotechnol 89 93 142 15217157
  2. [2]Villaverde A Carrió MM 2003 Protein aggregation in recombinant bacteria: biological role of inclusion bodies. Biotechnol Lett 25 85 95 14514038
  3. [3]Georgiou G Valax P 1999 Isolating inclusion bodies from bacteria. Methods Enzymol 309 48 58 10507015
  4. [5]Ventura S 2005 Sequence determinants of protein aggregation: tools to increase protein solubility. Microb Cell Fact 4 11 15847694 PMC1087874
  5. [6]Rinas U Bailey JE 1992 Protein compositional analysis of inclusion bodies produced in recombinant Escherichia coli. Appl Microbiol Biotechnol 37 609 614 1369400
  6. [7]Rudolph R 1996 Successful protein folding at industrial scale. Protein engineering: principles and practice 293 298 John Wiley and sons: New York
  7. [8]Vallejo LF Rinas U 2004 Strategies for the recovery of active proteins through refolding of bacterial inclusion body proteins. Microb Cell Fact 3 11 15345063 PMC517725
  8. [9]Panda AK 2003 Bioprocessing of therapeutic proteins from the inclusion bodies of Escherichia coli. Adv Biochem Eng/Biotechnol 85 43 93 12930093
  9. [10]Fink AL 1998 Protein aggregation: folding aggregates, inclusion bodies and amyloid. Fold Des 3 R9 23 9502314
  10. [11]Ventura S Villaverde A 2006 Protein quality in bacterial inclusion bodies. Trends Biotechnol 24 179 185 16503059
  11. [12]Przybycien TM Dunn JP Valax P Georgiou G 1994 Secondary structure characterization of β-lactamase inclusion bodies. Protein Eng 7 131 136 8140090
  12. [13]Oberg K Chrunyk BA Wetzel R Fink AL 1994 Native-like secondary structure in interleukin-1 β inclusion bodies by attenuated total reflectance FTIR. Biochemistry 33 2628 2634 8117725
  13. [14]Carrio M Gonzalez-Montalban N Vera A Villaverde A Ventura S 2005 Amyloid-like properties of bacterial inclusion bodies. J Mol Biol 347 1025 1037 15784261
  14. [15]Ignatova Z Krishnan B Bombardier JP Marcelino AM Hong J 2007 From the test tube to the cell: exploring the folding and aggregation of a β-Clam protein. Biopolymers 88 157 163 17206628 PMC2904568
  15. [16]Morell M Bravo R Espargaro A Sisquella X Aviles FX 2008 Inclusion bodies: specificity in their aggregation process and amyloid-like structure. Biochim Biophys Acta 1783 1815 1825 18619498
  16. [17]Doglia SM Ami D Natalello A Gatti-Lafranconi P Lotti M 2008 Fourier transform infrared spectroscopy analysis of the conformational quality of recombinant proteins within inclusion bodies. Biotechnol J 3 193 201 18213662
  17. [18]Wang L Maji SK Sawaya MR Eisenberg D Riek R 2008 Bacterial inclusion bodies contain amyloid-like structure. PLoS Biol 6 e195 18684013 PMC2494559
  18. [19]Speed MA Wang DIC King J 1996 Specific aggregation of partially folded polypeptide chains: the molecular basis of inclusion body composition. Nat Biotechnol 14 1283 1287 9631094
  19. [20]Margreiter G Messner P Caldwell KD Bayer K 2008 Size characterization of inclusion bodies by sedimentation field-flow fractionation. J Biotechnol 138 67 73 18760314 PMC4388406
  20. [21]Margreiter G Schwanninger M Bayer K Obinger C 2008 Impact of different cultivation and induction regimes on the structure of cytosolic inclusion bodies of TEM1-β-lactamase. Biotechnol J 3 1245 1255 18702088
  21. [22]Carrio MM Villaverde A 2001 Protein aggregation as bacterial inclusion bodies is reversible. FEBS Lett 489 29 33 11231008
  22. [23]Carrio MM Cubarsi R Villaverde A 2000 Fine architecture of bacterial inclusion bodies. FEBS Lett 471 7 11 10760503
  23. [24]Ami D Natalello A Gatti-Lafranconi P Lotti M Doglia SM 2005 Kinetics of inclusion body formation studied in intact cells by FT-IR spectroscopy. FEBS Letters 579 3433 3436 15949804
  24. [25]Garcia-Fruitos E Carrio MM Aris A Villaverde A 2005 Folding of a misfolding-prone β-galactosidase in absence of DnaK. Biotechnol Bioeng 90 869 875 15849697
  25. [26]Worrall DM Goss NH 1989 The formation of biologically active β- galactosidase inclusion bodies in Escherichia coli. Aust J Biotechnol 3 28 32 2485330
  26. [27]Garcia-Fruitos E Vazquez E Diez-Gil C Corchero JL Seras-Franzoso J 2012 Bacterial inclusion bodies: making gold from waste. Trends Biotechnol 30 65 70 22037492
  27. [28]Peternal S Komel R 2011 Active protein aggregates produced in Escherichia coli. Int J Mol Sci 12 8275 8287 22174663 PMC3233469
  28. [29]Jevsevar S Gaberc-Porekar V Fonda I Podobnik B Grdadolnik J 2005 Production of non-classical inclusion bodies from which correctly folded protein can be extracted. Biotechnol Prog 21 632 639 15801811
  29. [30]Peternal S Grdadolnik J Gaberc-Porekar V Komel R 2008 Engineering inclusion bodies for non denaturing extraction of functional proteins. Microb Cell Fact 7 34 19046444 PMC2630956
  30. [31]Carvajal P Gibert J Campos N Lopera O Barbera E 2011 Activity of maize transglutaminase over expressed in Escherichia coli inclusion bodies: An alternative to protein refolding. Biotechnol Prog 27 232 240 21312370
  31. [32]Patra AK Gahlay GK Reddy BV Gupta SK Panda AK 2000 Refolding, structural transition and spermatozoa-binding of recombinant bonnet monkey (Macaca radiata) zona pellucida glycoprotein-C expressed in Escherichia coli. Eur J Biochem 26 7075 7081 11106418
  32. [33]Umetsu M Tsumoto K Nitta S Adschiri T Ejima D 2005 Nondenaturing solubilization of β2 microglobulin from inclusion bodies by L-arginine. Biochem Biophys Res Commun 328 189 197 15670769
  33. [34]Singh SM Panda AK 2005 Solubilization and refolding of bacterial inclusion body proteins. J Biosci Bioeng 99 303 310 16233795
  34. [35]Kopito RR 2000 Aggresomes, inclusion bodies and protein aggregation. Trends Cell Biol 10 524 530 11121744
  35. [36]DeLellis RA Glenner GG Ram JS 1968 Histochemical observations on amyloid with reference to polarization microscopy. J Histochem Cytochem 16 663 665 4176941
  36. [37]Glenner GG Eanes ED Page DL 1972 The relation of the properties of Congo red-stained amyloid fibrils to the β-conformation. J Histochem Cytochem 20 821 826 4638557
  37. [38]Cooper JH 1974 Selective amyloid staining as a function of amyloid composition and structure. Histochemical analysis of the alkaline Congo red, standardized toluidine blue, and iodine methods. Lab Invest 31 232 238 4137921
  38. [39]LeVine H III 1997 Stopped-flow kinetics reveal multiple phases of thioflavin T binding to Alzheimer β (1–40) amyloid fibrils. Arch Biochem Biophys 342 306 316 9186492
  39. [40]Naiki H Higuchi K Hosokaw M Takeda T 1989 Fluorometric determination of amyloid fibrils in vitro using the fluorescent dye, thioflavin T1. Anal Biochem 177 244 249 2729542
  40. [41]Salnikova MS Middaugh CR Rytting JH 2008 Stability of lyophilized human growth hormone. Int J Pharm 358 108 113 18394830
  41. [42]Ward KR Adams GD Alpar HO Irwin WJ 1999 Protection of the enzyme L-asparaginase during lyophilisation-a molecular modeling approach to predict required level of lyoprotectant. Int J Pharm 187 153 162 10502621
  42. [43]Singh SM Sharma A Upadhyay AK Singh A Garg LC 2012 Solubilization of inclusion body proteins using n-propanol and its refolding into bioactive form. Protein Expr Purif 81 75 82 21964443
  43. [44]Wriston JC Jr 1985 Asparaginase. Methods Enzymol 113 608 618 3911011

Cited by

Sorted by citations

Authors
Upadhyay, A. K., Murmu, A., Singh, A., & Panda, A. K.
Affiliations
  • Baylor College of Medicine (US)
  • National Institute of Immunology (IN)
Corresponding author
Panda, Amulya K.
ORCID
Venue
PLoS ONE
Publisher
Public Library of Science (PLoS)
Published
2012-03-29
Volume
7
Issue
3
Pages
e33951
ISSN
1932-6203 (ISSN-L)
DOI
10.1371/journal.pone.0033951
PMC
PMC3315509
OpenAlex
W2160637206
Cited by
118
References
0
Citations by year
2026: 9 · 2025: 5 · 2024: 2 · 2023: 5 · 2022: 12 · 2021: 8 · 2020: 12 · 2019: 9 · 2018: 4 · 2017: 11 · 2016: 4 · 2015: 10 · 2014: 16 · 2013: 9 · 2012: 2
FWCI
4.76
Type
journal article
Language
en
Open access
Licence
Locations
  • PLoS ONE (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)
Funders
Jawaharlal Nehru University
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Protein purification and stability
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Heat shock proteins research
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Protein Structure and Dynamics
Keywords
inclusion bodies, protein aggregation kinetics, recombinant protein expression, human growth hormone, L-asparaginase, urea solubilization, protein refolding, proteinase K resistance, β-sheet content, hydrophobic interactions, Escherichia coli
MeSH terms
Amyloid, Asparaginase, Bacterial Proteins, Inclusion Bodies, Escherichia coli, Humans, Kinetics, Protein Binding, Recombinant Proteins, Solubility, Growth Hormone, Time Factors, Endopeptidase K, Proteolysis

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