Inclusion body

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. 

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). 

Polymyositis and Dermatomyositis Syndromes Polymyositis and Dermatomyositis Associated with Malignancy Inclusion Body Myositis (IBM)... 

In nature, there are several sources of enzymes that are capable of catalysing the hydrolysis of PHB. The polymer itself is produced by bacteria and occurs in cells as discrete inclusion bodies. These bodies contain the necessary enzymes for degrading the polymer, preventing its build-up in the cell. As well as this, there are numerous bacteria and fungi, many of which are found in the soil, that are capable of secreting the necessary enzymes outside their cell walls, and thus of iiufiating degradation of PHB. 

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. 

Hematological Methods. Hematological analyses can Include the determination of the total hemoglobin concentration (In g%), the packed cell volume (PCV In %), the red blood cell count (In 10 /mm ) and reticulocytes count (In %), calculation of the red cell Indices, examination of a blood film, tests to demonstrate the presence of Inclusion bodies and of sickle cells, tests to evaluate the distribution of fetal hemoglobin (Hb-F) Inside the red cells, the red cell osmotic fragility, the concentration of serum Iron (SI), total Iron binding capacity (TIBC), and the survival time of the red cells. Details of all... 

A search for Heinz bodies Is helpful In the detection of an unstable hemoglobin, of a-thalassemia, homozygous 3-thalas-semla and related abnormalities, because hemoglobin often precipitates In the red cells of patients with one of these disorders. Inclusion bodies may consist of precipitated unstable hemoglobin, of 3 chalns (In a-thalassemia), or of a chains (In thalassemia) ... 

Cells containing Hb-F are densely stained with erythrosln and cells with Hb-A appear as ghost cells Intermediate cells are stained more or less pink. Reticulocytes with Hb-A sometimes resemble Intermediate cells and may also show some Intracellular granulation. Inclusion bodies are visible In eluted cells as compact particles of differing sizes. Figure 10 gives some examples. The method Is Ideally suited to demonstrate the presence of newborn red cells In the maternal circulation. The method Is also widely used for the evaluation of the distribution of Hb-F within red cells mainly to differentiate between the HPFH condition and the 3 or 36 thalassemias. Evaluation of F cell smears In such cases Is difficult the term "equal distribution" usually Indicates the presence of Hb-F In each red cell but not necessarily In the same amount. 

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... 

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. 

Muller, C. and Rinas, U., Renaturation of heterodimeric platelet-derived growth factor from inclusion bodies of recombinant Escherichia coli using size-exclusion chromatography, /. Chromatogr. A, 855, 203, 1999. 

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... 

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