Abstract
Heterologous expression has long been used for the efficient production of proteins and enzymes as it offers significant advantages over purification of proteins from their native organisms. When first established, great efforts have been made to heterologously express proteins with high yields in the soluble fraction, hence, avoiding protein aggregation. In recent decades, however, it has been shown that the formation of aggregates (inclusion bodies; IBs) can be beneficial. To recover active protein, however, proteins should have been refolded from IBs after purification. The discovery that IBs themselves can also be active has revolutionized the entire protein production field. Therefore, several approaches have been described to generate catalytically active IBs during heterologous expression. Since several extrinsic and intrinsic factors such as protein structure and toxicity, pH and temperature of expression, and the used media might influence the formation of IBs, it is time and material consuming to use shake flask to examine and optimize different expression conditions. However, by using multi-well plates, it is possible to rapidly develop an efficient protocol for the expression of catalytically active IBs in a rational approach. The presented protocol was used for the heterologous expression of a 5′-adenosine monophosphate phosphorylase which forms catalytically active aggregates during expression in E. coli.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
García-Fruitós E (2010) Inclusion bodies: a new concept. Microb Cell Fact 9:80
Garcı E, Corchero L, Seras-franzoso J et al (2012) Bacterial inclusion bodies: making gold from waste. Trends Biotechnol 30:65–70
Rinas U, Garcia-Fruitós E, Uitós E et al (2017) Bacterial inclusion bodies: discovering their better half. Trends Biochem Sci 42:726–737
Gifre-Renom L, Seras-Franzoso J, Rafael D et al (2020) The biological potential hidden in inclusion bodies. Pharmaceutics 12:157
Wang L (2009) Towards revealing the structure of bacterial inclusion bodies. Prion 3:139
Singh A, Upadhyay V, Singh A et al (2020) Structure-function relationship of inclusion bodies of a multimeric protein. Front Microbiol 11:876
Krauss U, Jäger VD, Diener M et al (2017) Catalytically-active inclusion bodies—Carrier-free protein immobilizates for application in biotechnology and biomedicine. J Biotechnol 258:136–147
Jäger VD, Lamm R, Küsters K et al (2020) Catalytically-active inclusion bodies for biotechnology—general concepts, optimization, and application. Appl Microbiol Biotechnol 104:7313–7329
Ventura S (2005) Sequence determinants of protein aggregation: tools to increase protein solubility. Microb Cell Fact 4:1–8
Rodríguez-Bolaños M, Miranda-Astudillo H, Pérez-Castañeda E et al (2020) Native aggregation is a common feature among triosephosphate isomerases of different species. Sci Rep 10:1–14
Ventura S, Villaverde A (2006) Protein quality in bacterial inclusion bodies. Trends Biotechnol 24:179–185
Villaverde A, Carrió MM (2003) Protein aggregation in recombinant bacteria: biological role of inclusion bodies. Biotechnol Lett 25:1385–1395
Šiurkus J, Neubauer P (2011) Reducing conditions are the key for efficient production of active ribonuclease inhibitor in Escherichia coli. Microb Cell Fact 10:31
Ukkonen K, Veijola J, Vasala A et al (2013) Effect of culture medium, host strain and oxygen transfer on recombinant Fab antibody fragment yield and leakage to medium in shaken E. coli cultures. Microb Cell Fact 12:1–14
Kamel S, Walczak MC, Kaspar F et al (2021) Thermostable adenosine 5′-monophosphate phosphorylase from Thermococcus kodakarensis forms catalytically active inclusion bodies. Sci Rep 11:1–9
Diener M, Kopka B, Pohl M et al (2016) Fusion of a coiled-coil domain facilitates the high-level production of catalytically active enzyme inclusion bodies. ChemCatChem 8:142–152
Jäger VD, Kloss R, Grünberger A et al (2019) Tailoring the properties of (catalytically)-active inclusion bodies. Microb Cell Fact 18:1–20
Küsters K, Pohl M, Krauss U et al (2021) Construction and comprehensive characterization of an Ec LDCc – CatIB set — varying linkers and aggregation inducing tags. Microb Cell Fact. 20(1):49
Vasala A (2012) Cultivation plate system and method for the improved detection of microorganisms which contaminate food products. EP2476745A1
Neubauer P, Vasala A, Golson RK (2014) Methods for the supply of growth components to cell cultures. WO2009147200A3
Neubauer P, Neubauer A, Vasala A (2010) Enzyme based fed-batch technique in liquid cultures. EP2403936A1
Šiurkus J, Panula-Perälä J, Horn U et al (2010) Novel approach of high cell density recombinant bioprocess development: optimisation and scale-up from microlitre to pilot scales while maintaining the fed-batch cultivation mode of E. Coli Cultures 9:1–17
Szeker K, Niemitalo O, Casteleijn MG et al (2010) High-temperature cultivation and 5’ mRNA optimization are key factors for the efficient overexpression of thermostable Deinococcus geothermalis purine nucleoside phosphorylase in Escherichia coli. J Biotechnol 156:268–274
Rudolph R, Böhm G, Lilie H et al (1997) Folding proteins. In: Creighton TE (ed) Protein function: a practical approach. IRL Press at Oxford University Press, Oxford, pp 57–99
Acknowledgments
The authors would like to thank Miriam C. Walczak for the optimization work done on the IB purification and quantification. We are grateful for the support of Sarah Kamel by central innovation program for small- and medium-sized enterprise (ZIM).
Conflicts of Interest
Anke Kurreck is CEO and Peter Neubauer is a member of the advisory board of BioNukleo GmbH. Julia Schollmeyer is and Sarah Kamel was a scientific researcher at the biotech company BioNukleo GmbH. The authors have no other relevant affiliations or financial interests in or financial conflicts with the subject matter or materials discussed in the manuscript apart from those disclosed.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2023 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Kamel, S., Schollmeyer, J., Kurreck, A., Neubauer, P. (2023). Optimization of Inclusion Body Formation and Purification in Multi-well Plates. 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_8
Download citation
DOI: https://doi.org/10.1007/978-1-0716-2930-7_8
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