Inclusion Body Processing 4.0
CD Laboratory for Inclusion Body Processing 4.0
Laboratory Head: Assoc Prof. Oliver Spadiut
Academic Partner: TU Wien
Industrial Partner: Boehringer Ingelheim RCV GmbH & Co KG
Escherichia coli is one of the most widely used hosts to produce recombinant proteins. Its well-known genetics, the great variety of expression systems and production strains and the low cultivation costs as well as short generation times and high product titers make it very attractive for basic research as well as manufacturing purposes (e.g., [1-5]). To date, more than 25% of all biopharmaceuticals are produced in E. coli [3, 6-8]. However, a common consequence of overproduction of recombinant protein in E. coli is the formation of insoluble product aggregates, called Inclusion Bodies (IBs) (Figure 1; e.g., [4, 7, 9-12]). Inclusion Bodies occur in the polar region of the bacterium and are characterized by a porous structure, spherical or rod-shaped appearance and a diameter of up to around 700-800 nm [11, 13-19].
Figure 1: Inclusion Bodies (IBs). A, TEM picture showing IBs in recombinant E. coli cells; B, SEM picture of isolated IBs on a filter. Both pictures were taken at TU Wien in 2020.
Until around 20 years ago, IBs were considered as non-functional waste products and various strategies were developed to reduce or completely avoid their formation (e.g., [11, 20-28]). However, approximately 80% of all recombinant proteins overexpressed in E. coli still form IBs . In the last 20 years, numerous studies have demonstrated that proteins enclosed in IBs can be biologically active making IB formation an attractive strategy to produce large amounts of recombinant protein (e.g., [18, 30-48]). Thus, several direct applications of bioactive IBs in biocatalysis, synthetic chemistry and biomedicine have been developed [16, 38-42, 49, 50]. These bioactive IBs are used as immobilized biocatalysts [40, 41, 51-53], scaffolds and functional materials in tissue engineering [54-59], targeted and non-targeted drug delivery systems [57, 60, 61] as well as depots of therapeutic proteins, called nanopills [62, 63]. The importance of bioactive protein aggregates has recently been underlined by the development of pull-down tags, which induce IB formation of otherwise soluble proteins via intermolecular aggregation (e.g., [40, 64-73]).
Besides the direct use of catalytically active IBs, the fast-emerging market of biosimilars in the biopharmaceutical industry has a high demand for recombinant therapeutic proteins. Therefore, to ensure steady medical supply and enable economic manufacturing, it is of the utmost importance to enhance yield, accelerate process development time and lower production costs. Considering these aspects, the production of recombinant biopharmaceuticals in form of IBs instead of soluble protein presents a highly attractive option with many advantages [7, 14, 19, 45, 46, 74-82], such as high product yield (more than 10 g IBs/L cultivation broth), high product purity in IBs (up to 95%), high mechanical and thermal stability of IBs, resistance of IBs to proteases, easy isolation of IBs due to differences in size and density compared to host cell proteins, IBs contain native and native-like secondary protein structures and presence of biological activity.
Still, the current challenges for state-of-the-art IB processes are:
- To create a universal platform strategy rather than case-by-case approaches
- To miniaturize and automate process development
- To establish alternatives to harsh solubilization (e.g., extraction of soluble product from IBs, mild solubilization  or combinatorial strategies)
- To reduce buffer/water consumption and vessel size in refolding processes and thus reduce the environmental footprint
- To generate process knowledge and understanding
- To digitalize and pave the way to Industry 4.0
Within this CD Laboratory entitled “Inclusion Body Processing 4.0” we aim to address the listed challenges of state of the-art IB based processes, trying to pave the way to an universal, economic platform process, using sophisticated analytical and processing methods.
Figure 2: Overview of the CD Laboratory „Inclusion Body Processing 4.0“.
- Jozala, A.F., et al., Biopharmaceuticals from microorganisms: from production to purification. Brazilian Journal of Microbiology, 2016. 47: p. 51-63.
- Baeshen, M.N., et al., Production of Biopharmaceuticals in E. coli: Current Scenario and Future Perspectives. J Microbiol Biotechnol, 2015. 25(7): p. 953-62.
- Walsh, G., Biopharmaceutical benchmarks 2018. Nat Biotechnol, 2018. 36(12): p. 1136-1145.
- Rosano, G.L., E.S. Morales, and E.A. Ceccarelli, New tools for recombinant protein production in Escherichia coli: A 5-year update. Protein Sci, 2019. 28(8): p. 1412-1422.
- Castineiras, T.S., et al., E-coli strain engineering for the production of advanced biopharmaceutical products. Fems Microbiology Letters, 2018. 365(15).
- Gupta, S.K. and P. Shukla, Microbial platform technology for recombinant antibody fragment production: A review. Crit Rev Microbiol, 2017. 43(1): p. 31-42.
- Singhvi, P., et al., Bacterial Inclusion Bodies: A Treasure Trove of Bioactive Proteins. Trends Biotechnol, 2020. 38(5): p. 474-486.
- Baeshen, M.N., et al., Production of Biopharmaceuticals in E. coli: Current Scenario and Future Perspectives. Journal of Microbiology and Biotechnology, 2015. 25(7): p. 953-962.
- Singh, A., et al., Protein recovery from inclusion bodies of Escherichia coli using mild solubilization process. Microbial cell factories, 2015. 14: p. 41-41.
- Pauk, J.N., et al., Advances in monitoring and control of refolding kinetics combining PAT and modeling. Applied Microbiology and Biotechnology, 2021. 105(6): p. 2243-2260.
- Bhatwa, A., et al., Challenges Associated With the Formation of Recombinant Protein Inclusion Bodies in Escherichia coli and Strategies to Address Them for Industrial Applications. Frontiers in Bioengineering and Biotechnology, 2021. 9.
- Baneyx, F. and M. Mujacic, Recombinant protein folding and misfolding in Escherichia coli. Nature Biotechnology, 2004. 22(11): p. 1399-1408.
- Bowden, G.A., A.M. Paredes, and G. Georgiou, Structure and morphology of protein inclusion bodies in Escherichia coli. Nature Biotechnology, 1991. 9(8): p. 725.
- Rinas, U., et al., Bacterial inclusion bodies: discovering their better half. Trends in biochemical sciences, 2017. 42(9): p. 726-737.
- Wang, L., Towards revealing the structure of bacterial inclusion bodies. Prion, 2009. 3(3): p. 139-145.
- Rinas, U., et al., Bacterial Inclusion Bodies: Discovering Their Better Half. Trends Biochem Sci, 2017. 42(9): p. 726-737.
- Kopito, R.R., Aggresomes, inclusion bodies and protein aggregation. Trends in Cell Biology, 2000. 10(12): p. 524-530.
- Rueda, F., et al., Production of functional inclusion bodies in endotoxin-free Escherichia coli. Applied Microbiology and Biotechnology, 2014. 98(22): p. 9229-9238.
- Roca-Pinilla, R., et al., Exploring the use of leucine zippers for the generation of a new class of inclusion bodies for pharma and biotechnological applications. Microb Cell Fact, 2020. 19(1): p. 175.
- Ki, M.R. and S.P. Pack, Fusion tags to enhance heterologous protein expression. Appl Microbiol Biotechnol, 2020. 104(6): p. 2411-2425.
- Fatima, K., F. Naqvi, and H. Younas, A Review: Molecular Chaperone-mediated Folding, Unfolding and Disaggregation of Expressed Recombinant Proteins. Cell Biochemistry and Biophysics, 2021. 79(2): p. 153-174.
- Schlieker, C., B. Bukau, and A. Mogk, Prevention and reversion of protein aggregation by molecular chaperones in the E-coli cytosol: implications for their applicability in biotechnology. Journal of Biotechnology, 2002. 96(1): p. 13-21.
- Paraskevopoulou, V. and F.H. Falcone, Polyionic Tags as Enhancers of Protein Solubility in Recombinant Protein Expression. Microorganisms, 2018. 6(2).
- Waugh, D.S., The remarkable solubility-enhancing power of Escherichia coli maltose-binding protein. Postepy Biochem, 2016. 62(3): p. 377-382.
- Duan, X., et al., Efficient production of aggregation prone 4-alpha-glucanotransferase by combined use of molecular chaperones and chemical chaperones in Escherichia coli. J Biotechnol, 2019. 292: p. 68-75.
- Pina, A.S., C.R. Lowe, and A.C.A. Roque, Challenges and opportunities in the purification of recombinant tagged proteins. Biotechnology Advances, 2014. 32(2): p. 366-381.
- Gopal, G.J. and A. Kumar, Strategies for the production of recombinant protein in Escherichia coli. Protein J, 2013. 32(6): p. 419-25.
- Correa, A. and P. Oppezzo, Overcoming the solubility problem in E. coli: available approaches for recombinant protein production. Methods Mol Biol, 2015. 1258: p. 27-44.
- Jurgen, B., et al., Quality control of inclusion bodies in Escherichia coli. Microb Cell Fact, 2010. 9: p. 41.
- Peternel, S. and R. Komel, Active protein aggregates produced in Escherichia coli. Int J Mol Sci, 2011. 12(11): p. 8275-87.
- Carratala, J.V., et al., Title: insoluble proteins catch heterologous soluble proteins into inclusion bodies by intermolecular interaction of aggregating peptides. Microb Cell Fact, 2021. 20(1): p. 30.
- Villaverde, A., et al., Functional protein aggregates: just the tip of the iceberg. Nanomedicine (Lond), 2015. 10(18): p. 2881-91.
- Martinez-Alonso, M., E. Garcia-Fruitos, and A. Villaverde, Yield, solubility and conformational quality of soluble proteins are not simultaneously favored in recombinant Escherichia coli. Biotechnol Bioeng, 2008. 101(6): p. 1353-8.
- Garcia-Fruitos, E., A. Aris, and A. Villaverde, Localization of functional polypeptides in bacterial inclusion bodies. Appl Environ Microbiol, 2007. 73(1): p. 289-94.
- Garcia-Fruitos, E., et al., Bacterial inclusion bodies: making gold from waste. Trends Biotechnol, 2012. 30(2): p. 65-70.
- Worrall, D.M. and N.H. Goss, The formation of biologically active beta-galactosidase inclusion bodies in Escherichia coli. Aust J Biotechnol, 1989. 3(1): p. 28-32.
- Tokatlidis, K., et al., High activity of inclusion bodies formed in Escherichia coli overproducing Clostridium thermocellum endoglucanase D. FEBS Lett, 1991. 282(1): p. 205-8.
- Jager, V.D., et al., Tailoring the properties of (catalytically)-active inclusion bodies. Microbial Cell Factories, 2019. 18.
- Jager, V.D., et al., A Synthetic Reaction Cascade Implemented by Colocalization of Two Proteins within Catalytically Active Inclusion Bodies. Acs Synthetic Biology, 2018. 7(9): p. 2282-2295.
- Jager, V.D., et al., Catalytically-active inclusion bodies for biotechnology-general concepts, optimization, and application. Applied Microbiology and Biotechnology, 2020. 104(17): p. 7313-7329.
- Kloss, R., et al., Tailor-made catalytically active inclusion bodies for different applications in biocatalysis. Catalysis Science & Technology, 2018. 8(22): p. 5816-5826.
- Kloss, R., et al., Catalytically active inclusion bodies of L-lysine decarboxylase from E. coli for 1,5-diaminopentane production. Scientific Reports, 2018. 8.
- Carrio, M., et al., Amyloid-like properties of bacterial inclusion bodies. Journal of Molecular Biology, 2005. 347(5): p. 1025-1037.
- Singh, A., et al., Structure-Function Relationship of Inclusion Bodies of a Multimeric Protein. Frontiers in Microbiology, 2020. 11.
- Hoffmann, D., et al., Reassessment of inclusion body-based production as a versatile opportunity for difficult-to-express recombinant proteins. Critical Reviews in Biotechnology, 2018. 38(5): p. 729-744.
- Slouka, C., et al., Perspectives of inclusion bodies for bio-based products: curse or blessing? Appl Microbiol Biotechnol, 2019. 103(3): p. 1143-1153.
- Garcia-Fruitos, E., Inclusion bodies: a new concept. Microb Cell Fact, 2010. 9: p. 80.
- Ramon, A., M. Senorale-Pose, and M. Marin, Inclusion bodies: not that bad. Front Microbiol, 2014. 5: p. 56.
- de Marco, A., et al., Bacterial inclusion bodies are industrially exploitable amyloids. FEMS Microbiol Rev, 2019. 43(1): p. 53-72.
- Corchero, J.L., et al., Recombinant protein materials for bioengineering and nanomedicine. Nanomedicine (Lond), 2014. 9(18): p. 2817-28.
- Gatti-Lafranconi, P., et al., Concepts and tools to exploit the potential of bacterial inclusion bodies in protein science and biotechnology. FEBS J, 2011. 278(14): p. 2408-18.
- Sans, C., et al., Inclusion bodies of fuculose-1-phosphate aldolase as stable and reusable biocatalysts. Biotechnol Prog, 2012. 28(2): p. 421-7.
- Han, G.H., et al., Leucine zipper-mediated targeting of multi-enzyme cascade reactions to inclusion bodies in Escherichia coli for enhanced production of 1-butanol. Metabolic Engineering, 2017. 40: p. 41-49.
- Seras-Franzoso, J., et al., Improving protein delivery of fibroblast growth factor-2 from bacterial inclusion bodies used as cell culture substrates. Acta Biomater, 2014. 10(3): p. 1354-9.
- Unzueta, U., et al., Engineering tumor cell targeting in nanoscale amyloidal materials. Nanotechnology, 2017. 28(1): p. 015102.
- Martinez-Miguel, M., et al., Stable anchoring of bacteria-based protein nanoparticles for surface enhanced cell guidance. J Mater Chem B, 2020. 8(23): p. 5080-5088.
- Seras-Franzoso, J., et al., Cellular uptake and intracellular fate of protein releasing bacterial amyloids in mammalian cells. Soft Matter, 2016. 12(14): p. 3451-60.
- Seras-Franzoso, J., et al., Bioadhesiveness and efficient mechanotransduction stimuli synergistically provided by bacterial inclusion bodies as scaffolds for tissue engineering. Nanomedicine (Lond), 2012. 7(1): p. 79-93.
- Loo, Y., et al., Self-Assembled Proteins and Peptides as Scaffolds for Tissue Regeneration. Advanced Healthcare Materials, 2015. 4(16): p. 2557-2586.
- Carratala, J.V., et al., In Vivo Bactericidal Efficacy of GWH1 Antimicrobial Peptide Displayed on Protein Nanoparticles, a Potential Alternative to Antibiotics. Pharmaceutics, 2020. 12(12).
- Liovic, M., et al., Inclusion bodies as potential vehicles for recombinant protein delivery into epithelial cells. Microbial Cell Factories, 2012. 11.
- Seras-Franzoso, J., et al., A nanostructured bacterial bioscaffold for the sustained bottom-up delivery of protein drugs. Nanomedicine (Lond), 2013. 8(10): p. 1587-99.
- Vazquez, E., et al., Functional inclusion bodies produced in bacteria as naturally occurring nanopills for advanced cell therapies. Adv Mater, 2012. 24(13): p. 1742-7.
- Nahalka, J. and B. Nidetzky, Fusion to a pull-down domain: a novel approach of producing Trigonopsis variabilisD-amino acid oxidase as insoluble enzyme aggregates. Biotechnol Bioeng, 2007. 97(3): p. 454-61.
- Nahalka, J., A. Vikartovska, and E. Hrabarova, A crosslinked inclusion body process for sialic acid synthesis. Journal of Biotechnology, 2008. 134(1-2): p. 146-153.
- Nahalka, J., D. Mislovicova, and H. Kavcova, Targeting lectin activity into inclusion bodies for the characterisation of glycoproteins. Mol Biosyst, 2009. 5(8): p. 819-21.
- Nahalka, J., Physiological aggregation of maltodextrin phosphorylase from Pyrococcus furiosus and its application in a process of batch starch degradation to alpha-D-glucose-1-phosphate. Journal of Industrial Microbiology & Biotechnology, 2008. 35(4): p. 219-223.
- Yang, X.F., M. Pistolozzi, and Z.L. Lin, New trends in aggregating tags for therapeutic protein purification. Biotechnology Letters, 2018. 40(5): p. 745-753.
- Achmuller, C., et al., N-pro fusion technology to produce proteins with authentic N termini in E-coli. Nature Methods, 2007. 4(12): p. 1037-1043.
- Cheng, X.W., et al., Expression and purification of antimicrobial peptide CM4 by N-pro fusion technology in E. coli. Amino Acids, 2010. 39(5): p. 1545-1552.
- Wellhoefer, M., et al., Autoprotease N-pro: Analysis of self-cleaving fusion protein. Journal of Chromatography A, 2013. 1304: p. 92-100.
- Wang, X., et al., Formation of active inclusion bodies induced by hydrophobic self-assembling peptide GFIL8. Microbial Cell Factories, 2015. 14.
- Wang, W.Y., et al., Change of the N-terminal codon bias combined with tRNA supplementation outperforms the selected fusion tags for production of human d-amino acid oxidase as active inclusion bodies. Biotechnology Letters, 2017. 39(11): p. 1733-1740.
- Humer, D. and O. Spadiut, Wanted: more monitoring and control during inclusion body processing. World J Microbiol Biotechnol, 2018. 34(11): p. 158.
- Zielinski, M., et al., Expression and purification of recombinant human insulin from E. coli 20 strain. Protein Expr Purif, 2019. 157: p. 63-69.
- Upadhyay, V., A. Singh, and A.K. Panda, Purification of recombinant ovalbumin from inclusion bodies of Escherichia coli. Protein Expr Purif, 2016. 117: p. 52-8.
- Przybycien, T.M., et al., Secondary structure characterization of beta-lactamase inclusion bodies. Protein Eng, 1994. 7(1): p. 131-6.
- Trinh, N.T.M., T.L. Thuoc, and D.T.P. Thao, Production of recombinant human G-CSF from non-classical inclusion bodies in Escherichia coli. Braz J Microbiol, 2021. 52(2): p. 541-546.
- Sarker, A., A.S. Rathore, and R.D. Gupta, Evaluation of scFv protein recovery from E-coli by in vitro refolding and mild solubilization process. Microbial Cell Factories, 2019. 18.
- Peternel, S., et al., Fragility and solubility of non-classical inclusion bodies. Journal of Biotechnology, 2005. 118: p. S25-S25.
- Jevsevar, S., et al., Production of nonclassical inclusion bodies from which correctly folded protein can be extracted. Biotechnology Progress, 2005. 21(2): p. 632-639.
- Kaur, J., A. Kumar, and J. Kaur, Strategies for optimization of heterologous protein expression in E. coli: Roadblocks and reinforcements. International journal of biological macromolecules, 2017.
Projektass. Dipl.-Ing. Dr.techn. Julian Kopp BSc
Phone: +43 1 58801 166485 Call Julian Kopp
Projektass. Dipl.-Ing. Mohamed Elshazly BSc
Phone: +43 1 58801 166479 Call Mohamed Elshazly
Projektass. Dipl.-Ing. Robert Klausser BSc
Phone: +43 1 58801 166487 Call Robert Klausser