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Optimization of inclusion body solubilization and renaturation of recombinant human growth hormone from Escherichia coli.

Lead-induced inclusion bodies in renal tubular cells of rats have been studied in vitro after isolation by differential centrifugation. The inclusion bodies are insoluble in physiological media but may be dissolved in denaturants like 6M urea and sodium deoxycholate. They contain about 40–50 μg of lead/mg protein, but only about 10% of this is tightly bound. They also contain calcium, iron, zinc, copper, and cadmium. The protein is rich in glutamic and aspartic acids, glycine and cystine. When dissolved in 6M urea, the protein migrates as a single band on acrylamide gel electrophoresis and has a molecular weight of 27,500. It is suggested that the inclusion bodies function as an intracellular depot of nondiffusible lead. Further studies have been directed toward finding a free, unaggregated lead-containing protein fraction. Nuclear proteins from kidneys of lead-toxic rats were separated into NaCl-, Tris-, and NaOH-soluble fractions and an insoluble acidic fraction. A quantitatively small lead-containing protein was found in the 0.14M NaCl fraction. Amino acid composition, electrophoretic mobility, molecular weight, and ability to bind lead are similar to those of insoluble inclusion body protein. The possible role of this soluble lead-binding protein in the formation of nuclear inclusion bodies is at present time not certain. These studies do suggest, however, that protein-bound lead in renal tubular cells may be partitioned between insoluble and nondiffusible morphologically discrete inclusion bodies and a soluble, extractable fraction which is presumably diffusable.

Identification of reproduction-related proteins and characterization of the protein disulfide isomerase A6 cDNA in ovaries of the giant tiger shrimp Penaeus monodon

Molecular characterization and expression profiles of cyclin A and cyclin B during ovarian development of the giant tiger shrimp Penaeus monodon

Studies were made on the occurrence rates and ultrastructures of intracyto-plasmic crystalline inclusion bodies in the parietal cells of fattened steers. The abomasum examined was collected from 90 Holstein-Friesian and 13 Japanese Black-hair steers approximately 20 months old. The occurrence rates were different between the two breeds. The inclusion body was detected in 100% of the Holstein-Friesian and in 53.8% of the Japanese Black-hair steers. Inclusion bodies appeared specifically in fattened steers. They might be induced by unphysiological conditions of the abomasum under fattening. Crystalline inclusion bodies were homogeneous, acidophilic, and rod-shaped in H-E stained sections. Electron microscopic studies were performed on 4 Holstein-Friesian steers. The inclusion bodies were rod-like, hexagonal or rhombic in shape, and electron dense, without outer membrane, and located in the cytoplasmic matrix. There was no relationship between inclusion bodies and cytoplasmic organelles. The inclusion bodies appeared to reveal rod shape in the longitudinal section, regular hexagonal shape in the cross section, and rhombic shape in the oblique section. The fundamental shape of the crystalline inclusion body was considered to be a regular hexagonal rod with hemispherical ends. One side of the hexagon was 1.1-2.0μ in length. The longitudinal length of the rod was about 13-23μ. The internal structure of the crystalline inclusion body consisted of regularly arranged micro-tubular structures, which were about 115-154 Å in diameter. The center-to-center distance between two adjacent tubuli was about 190-200Å. The cross sections of the tubuli were hexagonal. Three adjacent tubuli gathered to form a regular triangle.

Kurloff cells, characterized by a large inclusion body in ill-defined lymphoreticular cells, were induced in male guinea pigs by oestrogen administration. The inclusion body material was characterized by way of light- and electron microscopy and histochemical staining reactions. Nucleo-protein were not detected, but certian tests for amino acids were observed and, at the peripheral parts of the inclusion bodies, phospholipid tests were positive. However, forced proteolysis of lipid extractions failed to abolish the reactions for detection of carbohydrate-rich compounds in the inclusion bodies thus confirming that their main characteristics were those of glycoproteins. Series of histochemical tests demonstrated the presence of periodate-reactive 1, 2-glycol groups and sulphate radicals. Periodate-reactive material and sulphomucins represented two separate substances, as demonstrated by extractions and combined staining tests. Some characteristics common with chondroitin sulphates were observed but, on the other hand, the observed differences suggested a different type of sulphation. The inclusion body failed to show any hydrolytic, dehydrogenase or oxidative enzyme activities, thus being metabolically inactive, although the cells showed certain enzyme activities in their cytoplasm and cell organelles.

The efficiency of physical separation of inclusion bodies from cell debris is related to cell debris size and inclusion body release and both factors should be taken into account when designing a process. In this work, cell disruption by enzymatic treatment with lysozyme and cellulase, by homogenization, and by homogenization with ammonia pretreatment is discussed. These disruption methods are compared on the basis of inclusion body release, operating costs, and cell debris particle size. The latter was measured with cumulative sedimentation analysis in combination with membrane-associated protein quantification by SDS-PAGE and a spectrophotometric peptidoglycan quantification method. Comparison of the results obtained with these two cell debris quantification methods shows that enzymatic treatment yields cell debris particles with varying chemical composition, while this is not the case with the other disruption methods that were investigated. Furthermore, the experiments show that ammonia pretreatment with homogenization increases inclusion body release compared to homogenization without pretreatment and that this pretreatment may be used to control the cell debris size to some extent. The enzymatic disruption process gives a higher product release than homogenization with or without ammonia pretreatment at lower operating costs, but it also yields a much smaller cell debris size than the other disruption process. This is unfavorable for centrifugal inclusion body purification in this case, where cell debris is the component going to the sediment and the inclusion body is the floating component. Nevertheless, calculations show that centrifugal separation of inclusion bodies from the enzymatically treated cells gives a high inclusion body yield and purity.

Segmentation of viral genomes provides the potential for genetic exchange within coinfected cells. However, for this potential to be realized, coinfecting genomes must mix during the viral life cycle. The efficiency of reassortment, in turn, dictates its potential to drive evolution. The opportunity for mixing within coinfected cells may vary greatly across virus families, such that the evolutionary implications of genome segmentation differ as a result of core features of the viral life cycle. To investigate the relationship between viral replication compartments and genetic exchange, we quantified reassortment in mammalian orthoreovirus (reovirus). Reoviruses carry a 10-segmented, double-stranded RNA genome, which is replicated within proteinaceous structures termed inclusion bodies. We hypothesized that inclusions impose a barrier to reassortment. We quantified reassortment between wild-type (wt) and variant (var) reoviruses that differ by one nucleotide per segment. Studies of wt/var systems in both T1L and T3D backgrounds revealed frequent reassortment without bias toward particular genotypes. However, reassortment was more efficient in the T3D serotype. Since T1L and T3D viruses exhibit different inclusion body morphologies, we tested the impact of this phenotype on reassortment. In both serotypes, reassortment levels did not differ by inclusion morphology. Reasoning that the merging of viral inclusions may be critical for genome mixing, we then tested the effect of blocking merging. Reassortment proceeded efficiently even under these conditions. Our findings indicate that reovirus reassortment is highly efficient despite the localization of many viral processes to inclusion bodies, and that the robustness of this genetic exchange is independent of inclusion body structure and fusion. IMPORTANCE Quantification of reassortment in diverse viral systems is critical to elucidate the implications of genome segmentation for viral evolution. In principle, genome segmentation offers a facile means of genetic exchange between coinfecting viruses. In practice, there may be physical barriers within the cell that limit the mixing of viral genomes. Here, we tested the hypothesis that localization of the various stages of the mammalian orthoreovirus life cycle within cytoplasmic inclusion bodies compartmentalizes viral replication and limits genetic exchange. Contrary to this hypothesis, our data indicate that reovirus reassortment occurs readily within coinfected cells and is not strongly affected by the structure or dynamics of viral inclusion bodies. We conclude that the potential for reassortment to contribute to reovirus evolution is high.

Many types of commercially valuable recombinant proteins produced by fermentation are expressed at high levels in Escherichia coli. Often, high-level expression in the host results in the formation of insoluble inclusion bodies. The release of these intracellular inclusion bodies from E. coli following cell disruption is a requirement for further downstream recovery. The ability to discern between intact unruptured cells and granules released from broken cells can provide valuable information for improving recovery yields in downstream purification. This paper describes a rapid and sensitive cytometry-based method that allows the simultaneous measurement of intact heat-killed E. coli and inclusion bodies using staining with nucleic acid binding fluorochromes.

The DNA sequence encoding the duplicated 22 amino acid segment of Clostridium thermocellum endoglucanase CelD was fused to the 3'-terminus of the celC gene encoding C.thermocellum endoglucanase CelC. The presence of the duplicated segment endowed CelC with the capacity to form cytoplasmic inclusion bodies containing active enzyme when the hybrid gene was expressed in Escherichia coli. Inclusion body formation prevented proteolytic cleavage of the duplicated segment. The intact hybrid protein CelC-Cel'D was purified from inclusion bodies and characterized. In contrast to CelC, CelC-Cel'D was able to bind to CipA, a protein acting as a scaffolding component of the C.thermocellum cellulase complex (cellulosome). However, the catalytic properties of CelC-Cel'D were similar to those of CelC. These results suggest that foreign proteins tagged with the duplicated segment could be incorporated into the cellulosome in order to modify the enzymatic properties of the complex. The formation of inclusion bodies by proteins carrying the duplicated segment may also prove a convenient means of purifying cloned gene products that are sensitive to proteolytic degradation.

DnaK and DnaJ facilitated the folding process and reduced inclusion body formation of magnesium transporter CorA overexpressed in Escherichia coli

Various concentrations of isopropyl beta-D-thiogalactopyranoside (IPTG) were used to induce production of the enzyme penicillin G acylase by recombinant Escherichia coli harboring plasmid pQEA11. The plasmid pQEA11 carries a wild-type pga gene, which is under the control of the tac promoter and lacIq. At low IPTG concentrations (0.025-0.1 mM), enzyme activity increased with increasing IPTG concentrations. At higher IPTG concentrations (0.2 and 0.5 mM), enzyme activity declined progressively. Examination of induced recombinant E. coli cells by transmission electron microscopy showed the presence of only periplasmic inclusion bodies at low IPTG concentrations (up to 0.1 mM) and both periplasmic and cytoplasmic inclusion bodies at high IPTG concentrations (0.2 mM and 0.5 mM). Results from sodium dodecyl sulfate/polyacrylamide gel electrophoresis and immunoblots of whole-cell proteins, membrane proteins and inclusion body proteins in these cells indicated that cytoplasmic inclusion bodies constituted an accumulation of preproenzyme (i.e., precursor polypeptide containing a signal peptide) and that periplasmic inclusion bodies constituted an accumulation of proenzyme (i.e., precursor polypeptide lacking a signal peptide).

Plant Virus Inclusion Bodies

Polypeptides associated with inclusion bodies from leaves of turnip infected with cauliflower mosaic virus

Clinical studies have shown that the antioxidant vitamin E can slow the progression of Alzheimer’s disease (AD). Other antioxidants reported to affect cognitive function include ginkgo biloba, vitamin C, and lipoic acid. To examine the effects of combination antioxidant therapy (CAT) on longevity and neuropathology in mice, we supplemented the diet of ApoE-deficient mice with vitamin E, ginkgo biloba, pycnogenol, and ascorbyl palmitate. ApoE-deficient mice normally exhibit increased numbers of PAS-positive inclusion bodies with aging. However, supplementation with CAT resulted in a significant increase in life span and a marked reduction of inclusion body histopathology in the hippocampus. In addition, while untreated apoE-deficient mice exhibited increased levels of TUNEL staining, a marker of DNA fragmentation, supplementation with CAT resulted in a significant reduction in the levels of TUNEL staining. These findings suggest that oxidative mechanisms, perhaps related to neuronal apoptosis, are integral to inclusion body formation in aging mice. The association between the reduced number of apoptotic cells and the reduction in inclusion bodies may explain in part the increased longevity of mice fed CAT, and supports the contention that the combined actions of selected antioxidants may be therapeutically effective against neurodegenerative diseases.