Inclusion bodies are nuclear or cytoplasmic aggregates of stainable substances, usually proteins. They typically represent sites of viral multiplication in a bacterium or a eukaryotic cell and usually consist of viral capsid proteins. Inclusion bodies can also be hallmarks of genetic diseases, as in the case of Neuronal Inclusion bodies in disorders like Frontotemporal dementia and Parkinson's disease.
Inclusion bodies contain very little host protein, ribosomal components or DNA/RNA fragments. They often almost exclusively contain the over expressed protein and aggregation in inclusion bodies has been reported to be reversible. It has been suggested that inclusion bodies are dynamic structures formed by an unbalanced equilibrium between aggregated and soluble proteins of E. coli. There is a growing body of information indicating that formation of inclusion bodies occurs as a result of intracellular accumulation of partially folded expressed proteins which aggregate through non-covalent hydrophobic or ionic interactions or a combination of both.
Inclusion bodies are dense electron-refractile particles of aggregated protein found in both the cytoplasmic and periplasmic spaces of E. coli during high-level expression of heterologous protein. It is generally assumed that high level expression of non-native protein (higher than 2% of cellular protein) and highly hydrophobic protein is more prone to lead to accumulation as inclusion bodies in E. coli. In the case of proteins having disulfide bonds, formation of protein aggregates as inclusion bodies is anticipated since the reducing environment of bacterial cytosol inhibits the formation of disulfide bonds. The diameter of spherical bacterial inclusion bodies varies from 0.5–1.3 m and the protein aggregates have either an amorphous or paracrystalline nature depending on the localization. Inclusion bodies have higher density (~1.3 mg ml–1) than many of the cellular components, and thus can be easily separated by high-speed centrifugation after cell disruption. Inclusion bodies despite being dense particles are highly hydrated and have a porous architecture.
Inclusion bodies have a non-unit lipid membrane. Protein inclusion bodies are classically thought to contain misfolded protein. However, this has recently been contested, as green fluorescent protein will sometimes fluoresce in inclusion bodies, which indicates some resemblance of the native structure and researchers have recovered folded protein from inclusion bodies.
Mechanism of formation
When genes from one organism are expressed in another the resulting protein sometimes forms inclusion bodies. This is often true when large evolutionary distances are crossed: a cDNA isolated from Eukarya for example, and expressed as a recombinant gene in a prokaryote risks the formation of the inactive aggregates of protein known as inclusion bodies. While the cDNA may properly code for a translatable mRNA, the protein that results will emerge in a foreign microenvironment. This often has fatal effects, especially if the intent of cloning is to produce a biologically active protein. For example, eukaryotic systems for carbohydrate modification and membrane transport are not found in prokaryotes. The internal microenvironment of a prokaryotic cell (pH, osmolarity) may differ from that of the original source of the gene. Mechanisms for folding a protein may also be absent, and hydrophobic residues that normally would remain buried may be exposed and available for interaction with similar exposed sites on other ectopic proteins. Processing systems for the cleavage and removal of internal peptides would also be absent in bacteria. The initial attempts to clone insulin in a bacterium suffered all of these deficits. In addition, the fine controls that may keep the concentration of a protein low will also be missing in a prokaryotic cell, and overexpression can result in filling a cell with ectopic protein that, even if it were properly folded, would precipitate by saturating its environment.
Viral inclusion bodies
Examples of viral inclusion bodies in animals are Intracytoplasmic eosinophilic-
- Negri bodies in Rabies
- Guarnieri bodies in Small pox
- Henderson-Peterson bodies in Molluscum contagiosum
- Cowdry type A in Herpes simplex virus and Varicella zoster virus and Torres bodies in Yellow fever
- Cowdry type B in Polio
Both intranuclear and intracytoplasmic-
- Warthin finkeldey bodies in Measles
Examples of viral inclusion bodies in plants  include aggregations of virus particles (like those for Cucumber mosaic virus ) and aggregations of viral proteins (like the cylindrical inclusions of potyviruses ). Depending on the plant and the plant virus family these inclusions can be found in epidermal cells, mesophyll cells, and stomatal cells when plant tissue when properly stained .
Inclusion bodies in Erythrocytes
Normally a red blood cell does not contain inclusions in the cytoplasm. However, it may be seen because of certain hematologic disorders.
There are three kinds of erythrocyte inclusions:
- Developmental Organelles
- Howell-Jolly bodies: small, round fragments of the nucleus resulting from karyorrhexis or nuclear disintegration of the late reticulocyte and stain reddish-blue with Wright stain.
- Basophilic stipplings - this stipplings is either fine or coarse, deep blue to purple staining inclusion that appears in erythrocytes on a dried Wright stain.
- Pappenheimer bodies - are siderotic granules which are small, irregular, dark-staining granules that appear near the periphery of a young erythrocyte in a Wright stain.
- Polychromatophilic red cells - young red cells that no longer have nucleus but still contain some RNA.
- Cabot Rings - ring-like structure and may appear in erythrocytes in megaloblastic anemia or in severe anemias, lead poisoning, and in dyserythropoiesis, in which erythrocytes are destroyed before being released from the bone marrow.
- Abnormal Hemoglobin Precipitation
- Heinz bodies - round bodies, refractile inclusions not visible on a Wright stain film. It is best identified by supravital staining with basic dyes.
- Hemoglobin H Inclusions - alpha thalassemia, greenish-blue inclusion bodies appear in many erythrocytes after four drops of blood is incubated with 0.5mL of Brilliant cresyl blue for 20 minutes at 37°C.
- Protozoan Inclusion
Current problems with the isolation of proteins from bacterial inclusion bodies
70-80% of recombinant proteins expressed E. coli are contained in inclusion bodies (i.e., protein aggregates).(Yang, Zhong, et al. "Highly efficient production of soluble proteins from insoluble inclusion bodies by a two-step-denaturing and refolding method." PloS one 6.7 (2011): e22981.) The purification of the expressed proteins from inclusion bodies usually require two main steps: extraction of inclusion bodies from the bacteria followed by the solubilisation of the purified inclusion bodies. This is considered labour-intensive, time consuming and not cost effective.
- Cruts, M; Gijselinck, I, van der Zee, J, Engelborghs, S, Wils, H, Pirici, D, Rademakers, R, Vandenberghe, R, Dermaut, B, Martin, JJ, van Duijn, C, Peeters, K, Sciot, R, Santens, P, De Pooter, T, Mattheijssens, M, Van den Broeck, M, Cuijt, I, Vennekens, K, De Deyn, PP, Kumar-Singh, S, Van Broeckhoven, C (2006-08-24). "Null mutations in progranulin cause ubiquitin-positive frontotemporal dementia linked to chromosome 17q21.". Nature 442 (7105): 920–4. doi:10.1038/nature05017. PMID 16862115.
- Biochem Biophys Res Com 328(2005) 189-197
- Protein Eng 7(1994) 131-136
- Biochem Biophys Res Comm 312 (2003) 1383-1386
- Chapter 20 in: Mitchell, Richard Sheppard; Kumar, Vinay; Abbas, Abul K.; Fausto, Nelson. Robbins Basic Pathology. Philadelphia: Saunders. ISBN 1-4160-2973-7. 8th edition.