Skip to main content
SpringerLink
Log in

High-Throughput Expression of Inclusion Bodies on an Automated Platform

  • Protocol
  • First Online:
Inclusion Bodies

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2617))

Abstract

In bioprocesses, which target the production of recombinant proteins as inclusion bodies, the upstream process has a decisive influence on the downstream operations, especially regarding cell disruption, inclusion body purity and composition, and refolding yield. Therefore, optimization of the processes in fed-batch mode is a major issue, and screening for strains and process conditions are performed in highly labor, time and cost intensive shake flasks or multiwell plates. Thus, high-throughput experiments performed similar to the industrial operating conditions offer a possibility to develop efficient and robust upstream processes. We present here an automated platform for Escherichia coli fed-batch cultivations in parallelized minibioreactors. The platform allows execution of experiments under multiple conditions while allowing for real-time monitoring of critical process parameters and a controlled fermentation environment. By this, the main factors that affect yields and quality of inclusion bodies can be investigated, speeding up the development process significantly.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution

Subscribe and save

Springer+ Basic
$34.99 /Month
  • Get 10 units per month
  • Download Article/Chapter or eBook
  • 1 Unit = 1 Article or 1 Chapter
  • Cancel anytime
Subscribe now

Buy Now

Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 109.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 139.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 219.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

Similar content being viewed by others

References

  1. Marston FA (1986) The purification of eukaryotic polypeptides synthesized in Escherichia coli. Biochem J 240:1–12. https://doi.org/10.1042/bj2400001

    Article  CAS  Google Scholar 

  2. 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. https://doi.org/10.1002/biot.200800072

    Article  CAS  Google Scholar 

  3. Georgiou G, Valax P (1999) Isolating inclusion bodies from bacteria. Methods Enzymol 309:48–58. https://doi.org/10.1016/S0076-6879(99)09005-9

    Article  CAS  Google Scholar 

  4. Strandberg L, Enfors SO (1991) Factors influencing inclusion body formation in the production of a fused protein in Escherichia coli. Appl Environ Microbiol 57:1669–1674. https://doi.org/10.1128/aem.57.6.1669-1674.1991

    Article  CAS  Google Scholar 

  5. Bhatwa A, Wang W, Hassan YI, Abraham N, Li X (2021) Challenges associated with the formation of recombinant protein inclusion bodies in Escherichia coli and strategies to address them for industrial applications. Front Bioeng Biotechnol 9:1–18. https://doi.org/10.3389/fbioe.2021.630551

    Article  Google Scholar 

  6. Carrió MM, Cubarsi R, Villaverde A (2000) Fine architecture of bacterial inclusion bodies. FEBS Lett 471:7–11. https://doi.org/10.1016/S0014-5793(00)01357-0

    Article  Google Scholar 

  7. Singh A, Upadhyay V, Upadhyay AK, Singh SM, Panda AK (2015) Protein recovery from inclusion bodies of Escherichia coli using mild solubilization process. Microb Cell Fact 14:41. https://doi.org/10.1186/s12934-015-0222-8

    Article  CAS  Google Scholar 

  8. Ramón A, Señorale-Pose M, Marín M (2014) Inclusion bodies: not that bad... Front Microbiol 5:2010–2015. https://doi.org/10.3389/fmicb.2014.00056

  9. Baeshen MN, Al-Hejin AM, Bora RS, Ahmed MMM, Ramadan HAI, Saini KS, Baeshen NA, Redwan EM (2015) Production of biopharmaceuticals in E. coli: current scenario and future perspectives. J Microbiol Biotechnol 25:953–962. https://doi.org/10.4014/jmb.1412.12079

    Article  CAS  Google Scholar 

  10. Rosano GL, Ceccarelli EA (2014) Recombinant protein expression in Escherichia coli: advances and challenges. Front Microbiol 5:1–17. https://doi.org/10.3389/fmicb.2014.00172

    Article  Google Scholar 

  11. Teworte S, Malcı K, Walls LE, Halim M, Rios-Solis L (2022) Recent advances in fed-batch microscale bioreactor design. Biotechnol Adv 55:107888. https://doi.org/10.1016/j.biotechadv.2021.107888

    Article  CAS  Google Scholar 

  12. Haby B, Hans S, Anane E, Sawatzki A, Krausch N, Neubauer P, Cruz Bournazou MN (2019) Integrated robotic mini bioreactor platform for automated, parallel microbial cultivation with online data handling and process control. SLAS Technol 24:569. https://doi.org/10.1177/2472630319860775

    Article  CAS  Google Scholar 

  13. Janzen NH, Striedner G, Jarmer J, Voigtmann M, Abad S, Reinisch D (2019) Implementation of a fully automated microbial cultivation platform for strain and process screening. Biotechnol J 14:1800625. https://doi.org/10.1002/biot.201800625

    Article  CAS  Google Scholar 

  14. Velez-Suberbie ML, Betts JPJJ, Walker KL, Robinson C, Zoro B, Keshavarz-Moore E (2017) High-throughput automated microbial bioreactor system used for clone selection and rapid scale-down process optimization. Biotechnol Prog 15:1–11. https://doi.org/10.1002/btpr.2534

    Article  CAS  Google Scholar 

  15. Šiurkus J, Neubauer P (2011) Reducing conditions are the key for efficient production of active ribonuclease inhibitor in Escherichia coli. Microb Cell Factories 10:1–15. https://doi.org/10.1186/1475-2859-10-31

    Article  CAS  Google Scholar 

  16. Anane E, Sawatzki A, Neubauer P, Cruz-Bournazou MN (2019) Modelling concentration gradients in fed-batch cultivations of E. coli – towards the flexible design of scale-down experiments. J Chem Technol Biotechnol:94. https://doi.org/10.1002/jctb.5798

  17. Funke M, Buchenauer A, Schnakenberg U, Mokwa W, Diederichs S, Mertens A, Müller C, Kensy F, Büchs J (2010) Microfluidic biolector-microfluidic bioprocess control in microtiter plates. Biotechnol Bioeng 107:497–505. https://doi.org/10.1002/bit.22825

    Article  CAS  Google Scholar 

  18. Hemmerich J, Noack S, Wiechert W, Oldiges M (2018) Microbioreactor systems for accelerated bioprocess development. Biotechnol J 13:1–9. https://doi.org/10.1002/biot.201700141

    Article  CAS  Google Scholar 

  19. Krause M, Ukkonen K, Haataja T, Ruottinen M, Glumoff T, Neubauer A, Neubauer P, Vasala A (2010) A novel fed-batch based cultivation method provides high cell-density and improves yield of soluble recombinant proteins in shaken cultures. Microb Cell Factories 9:1–11. https://doi.org/10.1186/1475-2859-9-11

    Article  CAS  Google Scholar 

  20. Glazyrina J, Materne E-M, Dreher T, Storm D, Junne S, Adams T, Greller G, Neubauer P (2010) High cell density cultivation and recombinant protein production with Escherichia coli in a rocking-motion-type bioreactor. Microb Cell Factories 9:42. https://doi.org/10.1186/1475-2859-9-42

    Article  CAS  Google Scholar 

  21. Sundström H, Enfors SO (2008) Software sensors for fermentation processes. Bioprocess Biosyst Eng 31:145–152. https://doi.org/10.1007/s00449-007-0157-5

    Article  CAS  Google Scholar 

  22. Anane E, García ÁC, Haby B, Hans S, Krausch N, Krewinkel M, Hauptmann P, Neubauer P, Cruz Bournazou MN (2019) A model-based framework for parallel scale-down fed-batch cultivations in mini-bioreactors for accelerated phenotyping. Biotechnol Bioeng 116:2906–2918. https://doi.org/10.1002/bit.27116

    Article  CAS  Google Scholar 

  23. Chory EJ, Gretton DW, DeBenedictis EA, Esvelt KM (2021) Enabling high-throughput biology with flexible open-source automation. Mol Syst Biol. 17:1–10. https://doi.org/10.15252/msb.20209942

    Article  Google Scholar 

  24. Osthege M, Tenhaef N, Zyla R, Müller C, Hemmerich J, Wiechert W, Noack S, Oldiges M (2022) bletl – a Python package for integrating BioLector microcultivation devices in the design-build-test-learn cycle. Eng Life Sci:1–18. https://doi.org/10.1002/elsc.202100108

  25. Arnold C (2022) Cloud labs: where robots do the research. Nature 606:612–613. https://doi.org/10.1038/d41586-022-01618-x

    Article  CAS  Google Scholar 

  26. Barz T, Sommer A, Wilms T, Neubauer P, Cruz Bournazou MN (2018) Adaptive optimal operation of a parallel robotic liquid handling station. Elsevier B.V., pp 765–770. https://doi.org/10.1016/j.ifacol.2018.04.006

    Book  Google Scholar 

  27. Cruz Bournazou MN, Barz T, Nickel DB, Lopez DC, Glauche F, Knepper A, Neubauer P (2017) Online optimal experimental re-design in robotic parallel fed-batch cultivation facilities. Biotechnol Bioeng 114:610–619. https://doi.org/10.1002/bit.26192

    Article  CAS  Google Scholar 

  28. Sawatzki A, Hans S, Narayanan H, Haby B, Krausch N, Sokolov M, Glauche F, Riedel SL, Neubauer P, Cruz Bournazou MN (2018) Accelerated bioprocess development of endopolygalacturonase-production with saccharomyces cerevisiae using multivariate prediction in a 48 mini-bioreactor automated platform. Bioengineering. 5. https://doi.org/10.3390/bioengineering5040101

  29. Hans S, Haby B, Krausch N, Barz T, Neubauer P, Cruz-Bournazou MN (2020) Automated conditional screening of multiple Escherichia coli strains in parallel adaptive fed-batch cultivations. Bioengineering 7:145. https://doi.org/10.3390/bioengineering7040145

    Article  CAS  Google Scholar 

  30. Krausch N, Kim JW, Barz T, Lucia S, Groß S, Huber MC, Schiller SM, Neubauer P, Cruz Bournazou MN (2022) High-throughput screening of optimal process conditions using model predictive control. Authorea. 558010. https://doi.org/10.22541/au.164600498.85996662/v2

  31. Anane E, López CDC, Neubauer P, Cruz Bournazou MN (2017) Modelling overflow metabolism in Escherichia coli by acetate cycling. Biochem Eng J. 125:23–30. https://doi.org/10.1016/j.bej.2017.05.013

    Article  CAS  Google Scholar 

  32. Kusterer A, Krause C, Kaufmann K, Arnold M, Weuster-Botz D (2008) Fully automated single-use stirred-tank bioreactors for parallel microbial cultivations. Bioprocess Biosyst Eng 31:207–215. https://doi.org/10.1007/s00449-007-0195-z

    Article  CAS  Google Scholar 

  33. Newton J, Oeggl R, Janzen NH, Abad S, Reinisch D (2020) Process adapted calibration improves fluorometric pH sensor precision in sophisticated fermentation processes. Eng Life Sci 20:331–337. https://doi.org/10.1002/elsc.201900156

    Article  CAS  Google Scholar 

  34. Hoffmann F, Rinas U (2004) Stress induced by recombinant protein production in Escherichia coli. Adv Biochem Eng Biotechnol 89:73–92. https://doi.org/10.1007/b93994

    Article  CAS  Google Scholar 

  35. Neubauer P, Häggström L, Enfors S-O (1995) Influence of substrate oscillations on acetate formation and growth yield in Escherichia coli glucose limited fed-batch cultivations. Biotechnol Bioeng 47:139–146. https://doi.org/10.1002/bit.260470204

    Article  CAS  Google Scholar 

  36. Neubauer P, Lin HY, Mathiszik B (2003) Metabolic load of recombinant protein production: inhibition of cellular capacities for glucose uptake and respiration after induction of a heterologous gene in Escherichia coli. Biotechnol Bioeng 83:53–64. https://doi.org/10.1002/bit.10645

    Article  CAS  Google Scholar 

  37. Bhattacharya SK, Dubey AK (1995) Metabolic burden as reflected by maintenance coefficient of recombinant Escherichia coli overexpressing target gene. Biotechnol Lett 17:1155–1160. https://doi.org/10.1007/BF00128377

    Article  CAS  Google Scholar 

  38. Lin H, Hanschke R, Nicklisch S, Nietsche T, Jarchow R, Riemschneider S, Meyer S, Gupta A, Hecker M, Neubauer P (2001) Cellular responses to strong overexpression of recombinant genes in Escherichia Coli. Recomb Protein Prod Prokaryotic Eukaryot Cells Comp View Host Physiol.:1–19. https://doi.org/10.1007/978-94-015-9749-4_5

  39. Mears L, Stocks SM, Albaek MO, Sin G, Gernaey KV (2017) Application of a mechanistic model as a tool for on-line monitoring of pilot scale filamentous fungal fermentation processes—the importance of evaporation effects. Biotechnol Bioeng 114:589–599. https://doi.org/10.1002/bit.26187

    Article  CAS  Google Scholar 

  40. Kawohl M, Heine T, King R (2007) Model-based estimation and optimal control of fed-batch fermentation processes for the production of antibiotics. Chem Eng Process Process Intensif 46:1223–1241. https://doi.org/10.1016/j.cep.2006.06.023

    Article  CAS  Google Scholar 

  41. Waldraff W, King R, Gilles ED (1997) Optimal feeding strategies by adaptive mesh selection for fed-batch bioprocesses. Bioprocess Eng 17:221–227. https://doi.org/10.1007/s004490050378

    Article  CAS  Google Scholar 

Download references

Acknowledgments

The authors would like to thank Jonathan Poit, Frederick Steiner, and Nicolas Feuser for the design and printing of the stirrer caps. Annina Kemmer thanks Boehringer Ingelheim RCV GmbH & Co KG for financial support. This work was supported by the German Federal Ministry of Education and Research through the International Future Labs for Artificial Intelligence Program (Grant number 01DD20002A KIWI-biolab).

Author information

Author notes

  1. Annina Kemmer and Linda Cai contributed equally.

Authors and Affiliations

  1. Technische Universität Berlin, Institute of Biotechnology Chair of Bioprocess Engineering, Berlin, Germany

    Annina Kemmer, Linda Cai, M. Nicolas Cruz Bournazou & Peter Neubauer

  2. DataHow AG, Dübendorf, Switzerland

    M. Nicolas Cruz Bournazou

Authors
  1. Annina Kemmer
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Linda Cai
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. M. Nicolas Cruz Bournazou
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Peter Neubauer
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Peter Neubauer .

Editor information

Editors and Affiliations

  1. Institute of Chemical, Environmental and Bioscience Engineering, TU Wien, Vienna, Austria

    Julian Kopp

  2. Institute of Chemical, Environmental and Bioscience Engineering, TU Wien, Vienna, Austria

    Oliver Spadiut

Rights and permissions

Reprints and permissions

Copyright information

© 2023 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Kemmer, A., Cai, L., Cruz Bournazou, M.N., Neubauer, P. (2023). High-Throughput Expression of Inclusion Bodies on an Automated Platform. In: Kopp, J., Spadiut, O. (eds) Inclusion Bodies. Methods in Molecular Biology, vol 2617. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2930-7_3

Download citation

  • DOI: https://doi.org/10.1007/978-1-0716-2930-7_3

  • Published:

  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-2929-1

  • Online ISBN: 978-1-0716-2930-7

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation