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PreprintCited by 18Protein purification and stabilityHeat shock proteins researchProtein Structure and Dynamics

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Chaperones facilitate heterologous expression of naturally evolved putative de novo proteins

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Eicholt, Lars A., Aubel, Margaux, Berk, Katrin, Bornberg-Bauer, Erich, Lange, Andreas·2022·Protein Science
DOI 10.1101/2022.03.02.482622PMC PMC9278007

Preprint — published as 10.1002/pro.4371 · Paper page

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Abstract

Over the past decade, evidence has accumulated that new protein coding genes can emerge de novo from previously non-coding DNA. Most studies have focused on large scale computational predictions of de novo protein coding genes across a wide range of organisms. In contrast, experimental data concerning the folding and function of de novo proteins is scarce. This might be due to difficulties in handling de novo proteins in vitro , as most are predicted to be short and disordered. Here we propose a guideline for the effective expression of eukaryotic de novo proteins in Escherichia coli . We used 11 sequences from Drosophila melanogaster and 10 from Homo sapiens , that are predicted de novo proteins from former studies, for heterologous expression. The candidate de novo proteins have varying secondary structure and disorder content. Using multiple combinations of purification tags, E. coli expression strains and chaperone systems, we were able to increase the number of solubly expressed putative de novo proteins from 30 % to 62 %. Our findings indicate that the best combination for expressing putative de novo proteins in E.coli is a GST-tag with T7 Express cells and co-expressed chaperones. We found that, overall, proteins with higher predicted disorder were easier to express.

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FIGURE 1 (a) Mechanism of chaperone assisted protein folding after Thomas et al. 38 The nascent protein is bound by the DnaK/J complex and release is triggered by GrpE under ATP hydrolysis. After release, the protein is either correctly folded, degraded (proteolysis), or remains unfolded. The unfolded protein can either aggregate or bind to the GroEL/ES complex. GroEL/ES either releases the folded protein by ATP hydrolysis or the protein is degraded. (b) Overview of the workflow on de novo protein expression: We first selected candidate proteins from Drosophila melanogaster (11, including Atlas) and 10 from Homo sapiens from a pool of putative de novo genes for expression. The 21 sequences were codon optimized for E. coli and ordered from Twist. For expression, different tags (GST and His), different E. coli expression cells (star, T7), and different chaperones (GroEL and DnaK systems) were tested. The success of protein expression was verified by Western blot (WB) and mass spectrometry (MS)
FIGURE 1 (a) Mechanism of chaperone assisted protein folding after Thomas et al. 38 The nascent protein is bound by the DnaK/J complex and release is triggered by GrpE under ATP hydrolysis. After release, the protein is either correctly folded, degraded (proteolysis), or remains unfolded. The unfolded protein can either aggregate or bind to the GroEL/ES complex. GroEL/ES either releases the folded protein by ATP hydrolysis or the protein is degraded. (b) Overview of the workflow on de novo protein expression: We first selected candidate proteins from Drosophila melanogaster (11, including Atlas) and 10 from Homo sapiens from a pool of putative de novo genes for expression. The 21 sequences were codon optimized for E. coli and ordered from Twist. For expression, different tags (GST and His), different E. coli expression cells (star, T7), and different chaperones (GroEL and DnaK systems) were tested. The success of protein expression was verified by Western blot (WB) and mass spectrometry (MS)CC BY · PubMed Central
FIGURE 2 Percentage of disorder as calculated with IUPred2a. All candidate de novo proteins used for expression experiments ordered by their disorder level from the left to right. Unicolor bars belong to the unsuccessfully expressed proteins, striped bars to the successfully expressed ones
FIGURE 2 Percentage of disorder as calculated with IUPred2a. All candidate de novo proteins used for expression experiments ordered by their disorder level from the left to right. Unicolor bars belong to the unsuccessfully expressed proteins, striped bars to the successfully expressed onesCC BY · PubMed Central
FIGURE 3 Percentage of random coils, α‐helices, and β‐sheets predicted by Porter 5.0 for each de novo protein candidate. Left to right following increasing disorder level based on Figure 2
FIGURE 3 Percentage of random coils, α‐helices, and β‐sheets predicted by Porter 5.0 for each de novo protein candidate. Left to right following increasing disorder level based on Figure 2CC BY · PubMed Central
FIGURE 4 Western blots with anti‐His antibody. Boxes indicate the height of the target protein band: DM1 (34 kDa): highest solubilty without chaperones, then GroEL, then DnaK; highly soluble. DM2 (36 kDa): only insoluble, even with chaperones. DM3 (33 kDa): DnaK highest solubilty, then base, then GroEL; very soluble. DM4 (34 kDa): DnaK highest solubilty, then GroEl, then base; very insoluble. DM5 (39 kDa): GroEL only one with soluble fraction, runs a bit high. DM7 (36 kDa): Dnak highest solubilty, then base, then GroEL very soluble. DM8 (37 kDa): all similar, different expression levels, first base, then GroEL, then DnaK; more insoluble. Atlas (20 kDa): GroEL highest solubilty, nothing in base and DnaK
FIGURE 4 Western blots with anti‐His antibody. Boxes indicate the height of the target protein band: DM1 (34 kDa): highest solubilty without chaperones, then GroEL, then DnaK; highly soluble. DM2 (36 kDa): only insoluble, even with chaperones. DM3 (33 kDa): DnaK highest solubilty, then base, then GroEL; very soluble. DM4 (34 kDa): DnaK highest solubilty, then GroEl, then base; very insoluble. DM5 (39 kDa): GroEL only one with soluble fraction, runs a bit high. DM7 (36 kDa): Dnak highest solubilty, then base, then GroEL very soluble. DM8 (37 kDa): all similar, different expression levels, first base, then GroEL, then DnaK; more insoluble. Atlas (20 kDa): GroEL highest solubilty, nothing in base and DnaKCC BY · PubMed Central
FIGURE 5 Western blots with anti‐His antibody. Boxes indicate the height of the target protein band: HS2 (44 kDa): upper bands (lower are degraded protein or double bands) most in DnaK, then GroEL, then base; very insoluble. HS7 (50 kDa): GroEL best, then base, nothing in DnaK. Possible protein degradation; very soluble. HS8 (16 kDa): upper bands most in DnaK, then GroEL, then base; very insoluble. HS9 (42 kDa): upper bands (lower are degraded protein or double bands) most in DnaK, then GroEL, then base; very insoluble. HS10 (43 kDa): upper bands (lower are degraded protein or double bands) most in GroEL, then DnaK, then base; very insoluble
FIGURE 5 Western blots with anti‐His antibody. Boxes indicate the height of the target protein band: HS2 (44 kDa): upper bands (lower are degraded protein or double bands) most in DnaK, then GroEL, then base; very insoluble. HS7 (50 kDa): GroEL best, then base, nothing in DnaK. Possible protein degradation; very soluble. HS8 (16 kDa): upper bands most in DnaK, then GroEL, then base; very insoluble. HS9 (42 kDa): upper bands (lower are degraded protein or double bands) most in DnaK, then GroEL, then base; very insoluble. HS10 (43 kDa): upper bands (lower are degraded protein or double bands) most in GroEL, then DnaK, then base; very insolubleCC BY · PubMed Central
FIGURE 6 Fractions of soluble and insoluble expression for candidate de novo proteins with expression of GroEL or DnaK and without (base). Values are intensities of Western blot bands
FIGURE 6 Fractions of soluble and insoluble expression for candidate de novo proteins with expression of GroEL or DnaK and without (base). Values are intensities of Western blot bandsCC BY · PubMed Central

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Research works printed but not linked: 66

  1. 10.1038/nrg3053 · DOI · Paper page
  2. 10.1038/nrg.2016.78 · DOI
  3. 10.12688/f1000research.10079.1 · DOI
  4. 10.1371/journal.pgen.1008160 · DOI
  5. 10.1016/j.tig.2019.08.007 · DOI
  6. 10.1016/j.sbi.2020.11.010 · DOI
  7. 10.1101/2022.01.14.476368 · DOI
  8. 10.1002/9780470619902.ch1 · DOI
  9. 10.1002/tera.1420090224 · DOI
  10. 10.1016/j.sbi.2013.02.012 · DOI
  11. 10.7554/eLife.53500 · DOI
  12. 10.1534/genetics.105.050336 · DOI
  13. 10.1534/genetics.107.084491 · DOI
  14. 10.1186/1471-2164-14-117 · DOI
  15. 10.1098/rstb.2014.0332 · DOI
  16. 10.1016/j.tig.2015.02.007 · DOI
  17. 10.1038/s41559-018-0639-7 · DOI
  18. 10.1093/molbev/msx315 · DOI
  19. 10.1534/g3.119.400326 · DOI
  20. 10.1038/s41559-019-0822-5 · DOI · Paper page
  21. 10.1007/s00239-020-09939-z · DOI
  22. 10.1093/gbe/evaa194 · DOI
  23. 10.1101/2022.01.04.474757 · DOI
  24. 10.1093/molbev/msw284 · DOI
  25. 10.1038/s41467-021-21667-6 · DOI
  26. 10.1016/j.str.2017.09.006 · DOI
  27. 10.1038/nature08009 · DOI
  28. 10.1126/science.1169375 · DOI
  29. 10.1016/j.sbi.2009.08.003 · DOI
  30. 10.1016/j.jmb.2022.167462 · DOI
  31. 10.1038/nrg2808 · DOI
  32. 10.1038/s41598-017-15635-8 · DOI
  33. 10.1186/1475-2859-7-11 · DOI
  34. 10.1042/BST20150089 · DOI
  35. 10.1146/annurev-biochem-060815-014124 · DOI
  36. 10.1073/pnas.1510083112 · DOI
  37. 10.1038/nsmb.1394 · DOI
  38. 10.1007/BF02785589 · DOI
  39. 10.1002/j.1460-2075.1993.tb06097.x · DOI
  40. 10.1038/nchembio.455 · DOI
  41. 10.1146/annurev-biochem-060208-092442 · DOI
  42. 10.1038/nature20137 · DOI
  43. 10.1038/337044a0 · DOI
  44. 10.1371/journal.pgen.1009787 · DOI
  45. 10.1002/cpbi.99 · DOI
  46. 10.1093/nar/gky384 · DOI
  47. 10.1101/289033 · DOI
  48. 10.1038/s41598-019-48786-x · DOI
  49. 10.1093/molbev/msu286 · DOI
  50. 10.1101/2022.01.13.476251 · DOI
  51. 10.2174/138920306775474095 · DOI
  52. 10.1002/0471140864.ps0519s50 · DOI
  53. 10.3389/fmicb.2014.00172 · DOI · Paper page
  54. 10.1007/978-1-60761-913-0_14 · DOI
  55. 10.1073/pnas.1201380109 · DOI
  56. 10.1155/2014/176172 · DOI
  57. 10.1073/pnas.1312296111 · DOI · Paper page
  58. 10.1007/978-1-0716-0524-0_27 · DOI
  59. 10.1021/acs.biochem.0c00961 · DOI
  60. 10.1126/science.abj8754 · DOI · Paper page
  61. 10.1093/bioadv/vbab043 · DOI
  62. 10.1093/bioinformatics/btz670 · DOI · Paper page
  63. 10.1128/AEM.64.5.1694-1699.1998 · DOI
  64. 10.1128/AEM.66.3.884-889.2000 · DOI
  65. 10.1016/0378-1119(90)90336-P · DOI
  66. 10.1101/pdb.prot3944 · DOI

Metadata

Authors
Eicholt, L. A., Aubel, M., Berk, K., Bornberg-Bauer, E., & Lange, A.
Affiliations
University of Münster (DE)
Corresponding author
Lange, Andreas
ORCID
Venue
Protein Science
Publisher
openRxiv
Published
2022-03-02
Volume
31
Issue
8
Pages
e4371-e4371
ISSN
0961-8368 (ISSN-L)
DOI
10.1101/2022.03.02.482622
PMC
PMC9278007
OpenAlex
W4285395243
Cited by
18
References
2
Citations by year
2026: 2 · 2025: 2 · 2024: 4 · 2023: 8 · 2022: 2
FWCI
1.31
Type
journal article
Language
en
Open access
Licence
cc-by
Locations
  • Protein Science (publishedVersion, cc-by)
  • bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion, cc-by-nc-nd)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
Funders
  • Deutsche Forschungsgemeinschaft (GRK 2220, 281125614)
  • Volkswagen Foundation (98183)
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
heterologous expression, molecular chaperones, Escherichia coli expression, protein solubility, intrinsically disordered proteins, Drosophila melanogaster, human, protein folding
MeSH terms
Animals, Drosophila melanogaster, Escherichia coli, Eukaryotic Cells, Protein Structure, Secondary, Molecular Chaperones

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CoChat. (2026). Citation check: Chaperones facilitate heterologous expression of naturally evolved putative de novo proteins. https://app.cochat.ai/papers/10.1101/2022.03.02.482622
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Eicholt, L. A., Aubel, M., Berk, K., Bornberg-Bauer, E., & Lange, A. (2022). Chaperones facilitate heterologous expression of naturally evolved putative de novo proteins. Protein Science. https://doi.org/10.1101/2022.03.02.482622