Inclusion bodies, enemy or ally?

What are inclusion bodies?

Recombinant protein expression has revolutionised biochemical research, allowing the study of protein interaction and function. Here at Peak Proteins, we specialise in the expression and purification of recombinant proteins. The vast majority of proteins in nature are active as soluble species and as such, recombinant protein production aims to obtain soluble proteins to mimic their natural states. This is particularly important for understanding how a protein functions natively – through biochemical or structural studies.

All living organisms have evolved an elaborate mechanism to maintain protein homeostasis (Proteostasis). This involves coordination of transcription, translation, post-translational modification (PTM) and degradation. Misfolding of proteins occurs when this equilibrium is unbalanced. With recombinant protein production this can often happen when the rate of a recombinant protein expression exceeds the ability of the host cells to manage protein folding [1], [2]. Incorrect PTMs (important to achieve a native, biologically active conformation) [3], size, hydrophobicity and low complexity regions all contribute to whether a protein might fold incorrectly and this can be predicted to a certain degree [4].

Misfolded proteins have a tendency to aggregate as the hydrophobic residues buried in the native protein are exposed on its surfaces. Bacterial expression systems (particularly E. coli) are widely used for recombinant protein productions due to advantages such as fast growth rate, cost and scalability. In E. coli, aggregated protein tend to eventually accumulate in dense, submicron particles known as inclusion bodies (IBs). IB formation during recombinant expression is often considered to be a major hurdle in protein production. Once described as being like trying to unboil an egg.

How can inclusion bodies be avoided?

One way to minimising IB formation is to reduce the expression rate of the recombinant protein – “Less is more!”. This can include reducing the temperature [5] or reducing inducer concentrations. An example where we employed this strategy at Peak Proteins can be found in this case study.

Another way to aid solubility is by the addition of a protein tag. The addition of a larger, very soluble protein tag can avoid aggregation long enough for the protein of interest to fold. Finally, alternative expressions hosts can be used instead to enable better folding and correct PTM. We employ Bacterial, Insect cell and Mammalian expression at Peak Proteins to ensure the right system is used for each protein.

But it’s not all bad!

Using IBs for protein production

Proteins accumulating in IBs can generally reach higher levels in comparison to those produced in soluble form. IBs also offer the advantages of being highly pure and resistant to proteases. Toxic proteins can often be best expressed in IBs where their altered folding renders them inert. We have used this approach at Peak Proteins to purify a nuclease that is otherwise toxic to cells. Utilising IBs, however, relies on being able to solubilise and then refold the protein of interest. This is an unpredictable and highly variable procedure. A growing number of recombinant protein products are produced as IBs, which are then processed into soluble product, for example Human Insulin, human growth factor, G-CSF and Interferon alfacon-1 [6]. Our team at Peak Proteins has vast experience in solubilising and refolding different protein targets from inclusion bodies. Indeed our CEO was a pioneer in the use of “factorial design / design of experiments (DoE)” [7], an incredibly powerful approach to negotiate the vast array of options available for the successful folding of a protein. The DoE approach has evolved since then and we now can employ a statistical software package JMP , to design and evaluate our experiments.

Direct IB applications

Recent evidence indicates a number of recombinant proteins that precipitate in IBs acquire native confirmations and are not necessarily inactive [8] [9], with activities ranging between 11% [10] and almost 100% [11]. One way this can be exploited is the development of IB-based biocatalysts. Immobilised enzymes are commonly used in bio-industrial applications. IBs are mechanically stable, relatively pure, easily separated and porous; making them promising alternatives to current immobilised catalysts [12][13].

Another application of IBs is as a protein delivery vehicle or “nano-pill”. IBs have been demonstrated to be internalised by mammalian cells [14][15] and subsequent protein release has been verified [14][16] including in animal models [17].

In an approach directly contradictory to the above discussion of prevention of IB formation; fusion to an aggregate-prone domain can be used force soluble proteins into IBs. A number of these tags have been developed [18] allowing proteins of interest to be driven into aggregation.

These recent developments have led a once maligned biproduct to be rebranded as a promising biotechnological tool.

Our staff at Peak Proteins have considerable experience expressing and purifying a wide range of classes of protein. Should that be of interest to you and your research, please don’t hesitate to get in touch with us via our website or by email (info@peakproteins.com). We look forward to hearing from you.

References:

1. T. Gill, J.J. Valdes, W.E. Bentley, A Comparative Study of Global Stress Gene Regulation in Response to Overexpression of Recombinant Proteins in Escherichia coli, Metabolic Engineering, Volume 2, Issue 3, 2000, Pages 178-189 https://doi.org/10.1006/mben.2000.0148
2. Quantification of proteomic and metabolic burdens predicts growth retardation and overflow metabolism in recombinant Escherichia coli, Hong Zeng,Aidong Yang. First published: 03 February 2019 https://doi.org/10.1002/bit.26943
3. Gary Walsh, Post-translational modifications of protein biopharmaceuticals, Drug Discovery Today, Volume 15, Issues 17–18, 2010, Pages 773-780 https://doi.org/10.1016/j.drudis.2010.06.009
4. A Consensus Method for the Prediction of ‘Aggregation-Prone’ Peptides in Globular Proteins Antonios C. Tsolis,Nikos C. Papandreou,Vassiliki A. Iconomidou,Stavros J. Hamodrakas Published: January 10, 2013 https://doi.org/10.1371/journal.pone.0054175
5. Shmuel Cabilly, Growth at sub-optimal temperatures allows the production of functional, antigen-binding Fab fragments in Escherichia coli, Gene, Volume 85, Issue 2, 1989, Pages 553-557 https://doi.org/10.1016/0378-1119(89)90451-4
6. Manufacturing of recombinant therapeutic proteins in microbial systems, Klaus Graumann Dr. First published: 07 February 2006 https://doi.org/10.1002/biot.200500051
7. Tobbell DA, Middleton BJ, Raines S, Needham MR, Taylor IW, Beveridge JY, Abbott WM. Identification of in vitro folding conditions for procathepsin S and cathepsin S using fractional factorial screens. Protein Expr Purif. 2002 Mar;24(2):242-54. doi: 10.1006/prep.2001.1573. PMID: 11858719.
8. Volume 24, Issue 4, P179-185, April 01, 2006 Protein quality in bacterial inclusion bodies Salvador Ventura Antonio Villaverde DOI:https://doi.org/10.1016/j.tibtech.2006.02.007
9. Mar Carrió, Nuria González-Montalbán, Andrea Vera, Antonio Villaverde, Salvador Ventura, Amyloid-like Properties of Bacterial Inclusion Bodies, Journal of Molecular Biology, Volume 347, Issue 5, 2005, Pages 1025-1037 https://doi.org/10.1016/j.jmb.2005.02.030
10. The conformational quality of insoluble recombinant proteins is enhanced at low growth temperatures Andrea Vera,Nuria González-Montalbán,Anna Arís,Antonio Villaverde. First published: 29 September 2006 https://doi.org/10.1002/bit.21218
11. High activity of inclusion bodies formed in Escherichia coli overproducing Clostridium thermocellum endoglucanase D Kostas Tokatlidis,Prasad Dhurjati,Jacqueline Millet,Pierre Béguin,Jean-Paul Aubert https://doi.org/10.1016/0014-5793(91)80478-L
12. Enzymatic synthesis of sialylation substrates powered by a novel polyphosphate kinase (PPK3)† Jozef Nahálka and Vladimír Pätoprstýa https://doi.org/10.1039/B822549B
13. Fusion to a pull-down domain: a novel approach of producing Trigonopsis variabilisD-amino acid oxidase as insoluble enzyme aggregates Jozef Nahalka,Bernd Nidetzky First published: 06 November 2006 https://doi.org/10.1002/bit.21244
14. Functional Inclusion Bodies Produced in Bacteria as Naturally Occurring Nanopills for Advanced Cell Therapies Esther Vázquez,José L. Corchero,Joan F. Burgueño,Joaquin Seras-Franzoso,Ana Kosoy,Ramon Bosser,Rosa Mendoza,Joan Marc Martínez-Láinez,Ursula Rinas,Ester Fernández,Luis Ruiz-Avila,Elena García-Fruitós,Antonio Villaverde First published: 13 March 2012 https://doi.org/10.1002/adma.201104330
15. Cellular uptake and intracellular fate of protein releasing bacterial amyloids in mammalian cells Joaquin Seras-Franzoso, Alejandro Sánchez-Chardi, Elena Garcia-Fruitós, Esther Vázquezabc and  Antonio Villaverdeabc  https://doi.org/10.1039/C5SM02930A
16. Liovic, M., Ozir, M., Zavec, A.B. et al. Inclusion bodies as potential vehicles for recombinant protein delivery into epithelial cells. Microb Cell Fact 11, 67 (2012). https://doi.org/10.1186/1475-2859-11-67
17. Débora Torrealba, David Parra, Joaquin Seras-Franzoso, Eva Vallejos-Vidal, Daniel Yero, Isidre Gibert, Antonio Villaverde, Elena Garcia-Fruitós, Nerea Roher, Nanostructured recombinant cytokines: A highly stable alternative to short-lived prophylactics, Biomaterials, Volume 107, 2016, Pages 102-114, ISSN 0142-9612, https://doi.org/10.1016/j.biomaterials.2016.08.043
18. Bacterial Inclusion Bodies: Discovering Their Better Half Ursula Rinas Elena Garcia-Fruitós José Luis Corchero Esther Vázquez Joaquin Seras-Franzoso Antonio Villaverde Published: February 26, 2017 DOI:https://doi.org/10.1016/j.ti

Written by Martin Jennings