Protein inclusion bodies

Materials produced by crystalliferous bacilli which elicit a toxic response in susceptible insects may be separated into two types. The first type, the true toxins, include the crystalline protein inclusion body the parasporal body of Hannay (14)], a heat-stable, water-soluble exotoxin active against flies, a heat-stable, dialyzable water-soluble exotoxin, toxic to Lepidoptera on injection (23), and a heat-labile, water-soluble, filterable exotoxin, toxic toward larch sawfly larvae (Hymenoptera) which was reported by Smirnoff (31). 

Intracellular products can be present either as folded, soluble proteins or as dense masses of unfolded protein (inclusion bodies). For these products, it is first necessary to concentrate the cell suspension before effecting release of the product. Filtration can result in a suspension of cells that can be of any desired concentration up to 15 to 17 percent and that can be diafiltered into the desired buffer system. In contrast, the cell slurry that results from centrifugation will be that of... 

Kane, J.F. and Hartley, D.L. (1988) Formation of recombinant protein inclusion bodies in... 

It is well known that a large number of chemical substances, including toxic metals and metalloids such as arsenic, cadmium, lead, and mercury, cause cell injury in the kidney. With metal-induced neurotoxicity, factors such as metal-binding proteins, inclusion bodies, and cell-specific receptor-like proteins seem to influence renal injury in animals and humans. It is of interest to note that certain renal cell populations become the targets for metal toxicity, while others do not. In fact, the target cell populations handle the organic and common inorganic nephrotoxicants differently. ... 

Bowden, G.A., Paredes, A.M., and Georgiou, G. (1991). Structure and morphology of protein inclusion bodies in Escherichia coli. Bio/Technology 9, 725 730. 

Expression of proteins in E. coli often results in the formation of insoluble aggregates called inclusion bodies, probably comprising fully or partially unfolded proteins. Inclusion bodies are brought to their monomeric form by extraction with a denaturant (e.g., 8 M urea) under reducing conditions (e.g., 0.1 M cysteine). 

Rogl, H., Kosemund, K., Kiihlbrandt, W., CoDinson, I. (1998) Refolding of Escherischia coli produced membrane protein inclusion bodies immobilised by nickel chelating chromatography. FEES Letters, 432, 21—26. 

Whereas recombinant proteins produced as inclusion bodies in bacterial fermentations may be amenable to reversed-phase chromatography (42), the use of reversed-phase process chromatography does not appear to be widespread for higher molecular weight proteins. 

The methods involved in the production of proteins in microbes are those of gene expression. Several plasmids for expression of proteins having affinity tails at the C- or N-terminus of the protein have been developed. These tails are usefiil in the isolation of recombinant proteins. Most of these vectors are commercially available along with the reagents that are necessary for protein purification. A majority of recombinant proteins that have been attempted have been produced in E. Coli (1). In most cases these recombinant proteins formed aggregates resulting in the formation of inclusion bodies. These inclusion bodies must be denatured and refolded to obtain active protein, and the affinity tails are usefiil in the purification of the protein. Some of the methods described herein involve identification of functional domains in proteins (see also Protein engineering). 

Cell Disruption Intracellular protein products are present as either soluble, folded proteins or inclusion bodies. Release of folded proteins must be carefully considered. Active proteins are subject to deactivation and denaturation, and thus require the use of gentle conditions. In addition, due consideration must be given to the suspending medium lysis buffers are often optimized to promote protein stability and protect the protein from proteolysis and deactivation. Inclusion bodies, in contrast, are protected by virtue of the protein agglomeration. More stressful conditions are typically employed for their release, which includes going to higher temperatures if necessaiy. For native proteins, gentler methods and temperature control are required. 

B. thurigiensis is a common Gram-positive, spore-forming soil bacterium that produces inclusion bodies, microcrystalline clusters of many different proteins. These crystalline proteins, called 5-endotoxins, are the ion channel toxins that are sold commercially for pest control. Most such endotoxins are protoxins, which are inactive until cleaved to smaller, active proteins by proteases in the gut of a susceptible insect. One such crystalline protoxin. 

The 26 kDa protein synthesised by salt-adapted tobacco cells has been further characterised (Singh et al., 1987a). The protein makes up approximately 12% of the total cellular protein and has been resolved into two forms. These two forms have been designated osmotin 1 and osmotin II and occur in a 2 3 ratio. The forms are distinct with osmotin I soluble in an aqueous phase and osmotin II soluble in detergent. The proteins accumulate as inclusion bodies in the vacuole and are only sparsely distributed in the cytoplasm. 

FIG. 20-87 Illustration of a refolding process for a protein from inclusion body. 

Titchener-Hooker, N. J., Gritsis, D., Mannweiler, K., Olbrich, R., Gardiner, S. A. M., Fish, N. M., and Hoare, M., Integrated process design for producing and recovering proteins from inclusion bodies, BioPharm, July/Aug., 34,1991. 

To this list of protein misfolding diseases can be added rare familial amyloidoses in which the mutated proteins have the classic amyloid fibril congophilic birefringence and cross-(3-sheet structure (Table 3). Many of these deposits have an impact on the central nervous system (TTR, cystatin, lysozyme) as well as on other organ systems. A newly described disease, familial British dementia, is associated with the deposition of Abri, a 34 amino acid, 4 kDa peptide cleaved from a 277 amino acid precursor sequence, the last 10 amino acids of which are not normally translated [52]. Familial encephalopathy with neuroserpin inclusion bodies (FENIB) is... 

Histopathological changes were noted in the kidneys of rats administered lead acetate in the drinking water for 76 weeks at lower doses (Roller et al. 1985). These changes, which were observed at a dose of 37 mg/kg/day, a dose much lower than that used by Vyskocil et al. (1989), included necrotic and dilated cortical tubules, tubular protein casts, areas with large nuclei and fibrous connective tissue, and large intranuclear inclusion bodies in the enlarged epithelial cells of the cortex near the cortical-medullary junction. 

---