• Journal of Microbiology and Biotechnology
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• v.6 no.4
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• pp.225-231
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• 1996

The production of recombinant proteins in Escherichia coli often leads to the formation of an intracellular inclusion body. Key process steps that can determine the economics of large-scale protein production from inclusion bodies are fermentation, inclusion body recovery, and protein refolding. Compared with protein refolding and fermentation, inclusion body recovery has received scant research attention. Nevertheless, it can control the final product yield and hence process cost for some products. Optimal separation of inclusion bodies and cell debris can also aid subsequent operations by removing contaminant particulates that foul chromatographic resins and contain antigenic pyrogens. In this review, the properties of inclusion bodies and cellular debris are therefore examined. Attempts to optimise the centrifugal separation of inclusion bodies and debris are also discussed.

• KSBB Journal
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• v.16 no.1
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• pp.11-14
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• 2001

Techniques for protein refolding from inclusion body are discussed in view of its engineering application to large scale protein purification. Among the techniques, dilution and dialysis are mainly utilized due to simple operation. Membrane reactor, gel filtration chromatography, and continuous tank operation are emerging tools for their process-scale possibility in refolding. Reaction engineering approaches could be used to analyze the kinetic behaviour in the process scale refolding reactor. The kinetic analysis is helpful in the optimization of refolding yield in the refolding reactor.

• KSBB Journal
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• v.6 no.1
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• pp.111-114
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• 1991

The effects of the precursor processing inhibitor, carbonylcyanide-chlorophenyl hydrazone(CCCP), are investigated on the production of soluble ${\beta}-lactamase$ the formation of the inclusion body when ${\beta}-lactamase$ is overproduced by induction with isopropyl thiogalactoside(IPTG). When cells are treated by CCCP, more soluble ${\beta}-lactamase$ is produced. In this case, no difference in the amount of inclusion body is observed.

• Korean Journal of Veterinary Research
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• v.22 no.1
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• pp.37-52
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• 1982

This study was taken to clarify the histopathological changes of pigs naturally infected with hog cholera. Microscopic observations of the necrotic lesion and inclusion body in the lymphoid organs were carried out in the natural cases of hog cholera and experimental cases inoculated with ALD virus and isolated virus strains. Electron microscopic findings of the intranuclear inclusion bodies in the reticular cell of spleen and lymph node were also observed in the experimental cases. The results obtained are as follow, As the histological findings necrosis of lymphoid organs was observed mainly in the lymph follicle. The necrotic lymphoid organs were found to contain 35.0% in the natural and 37.5% in the experimental cases. Intranuclear inclusion bodies were found mainly in the reticular cells of lymphoid organ, the epithelium of bronchiole and alevolus, and the vascular endothelium of brain. These inclusion bodies were seen in 40.0% of the natural cases and all of the experiment. The inclusion body was appeared to compose of activated nucleoli and chromatin granules (interchromatin and perichromatin) by electron microscopy.

• Korean Journal of Veterinary Research
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• v.21 no.1
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• pp.41-43
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• 1981

The occurrence of inclusion body hepatitis was confirmed for the first time in Korea from chickens submitted for diagnosis to this Institute from a farm located in the vicinity of Anyang. The chickens showed no specific clinical signs except for moderate anemic conditions. At autopsy, livers were swollen and mottled with numerous stellate subcapsular hemorrhages and necrotic foci. Severe nephritis and catarrhal enteritis were also seen. The most notable microscopic changes were seen in the liver. These included eosinophilic intra-nuclear inclusion bodies in the hepatocyes, massive hemorrhages and necrosis and fatty changes in the liver parenchema.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.44 no.5
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• pp.560-564
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• 2011

Two feeding experiments were conducted to investigate the effects of dietary inclusion of genetically modified (GM) soybean and corn on the growth performance, feed utilization and body composition of juvenile abalone Haliotis discus hannai. Four isonitrogenous (31% crude protein) and isolipidic (6% crude lipid) diets (designated as nGM-soya, GM-soya, nGM-corn and GM-corn) were formulated to contain 20% non-GM (nGM) and GM soya and corn. Fifty juvenile abalone (initial body weight, 2.0 g) were distributed in each 50 L tank in a flow-through system. Each experimental diet was fed to duplicate groups of abalone to satiation once a day for 10 weeks. No effects of GM feedstuffs on survival were observed. Dietary inclusion of GM feedstuffs did not affect either growth performance or feed utilization of abalone. Body composition was not altered by the inclusion of GM feedstuffs. These results indicate that dietary inclusion of GM soybean and corn could have no effect on the growth performance and body composition of juvenile abalone. Further studies to investigate the effects of GM feedstuffs on transgenic fragment residues in ambient environments and in animals are necessary for the safe use of such ingredients in aquaculture.

• BMB Reports
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• v.30 no.2
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• pp.144-149
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• 1997

An expression vector was constructed containing the gene encoding diphtheria toxin A (DTA) which was placed after a T7 promoter. Cytoplasmic expression of the DTA gene resulted in the formation of an insoluble inclusion body. The inclusion body was collected after the complete lysis of the cell, and subsequent washing with 0.1% Triton X-100 released 16~30% of DTA protein from the inclusion body along with other contaminating proteins. The released DTA protein was purified by dialysis. The remaining pellet was dissolved in 8 M urea containing 5% ${\beta}-mercaptoethanol$, and the denatured DTA was renatured by the dilution-dialysis method. The total yield was 35%, and about 5 mg DTA was obtained from 1 L culture. The DTA protein has a free sulfhydryl group exposed to the protein surface, and was shown to have a tendency to dimerize through disulfide formation in the absence of ${\beta}-mercaptoethanol$. The utility of the sulfhydryl group was tested for the construction of recombinant toxins.

• Animal cells and systems
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• v.16 no.6
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• pp.455-461
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• 2012

Amongst the various antimicrobial peptides, lysozyme plays a central role in initiating and maintaining the antibacterial defense response of insect. Here we propose the biosynthesis and refolding of recombinant lysozyme in Escherichia coli expressed in inclusion body form. The Agrius lysozyme gene was amplified using gene specific primers and then ligated into the pGEX-4T-1 vector, which contained the glutathione S-transferase (GST) gene as a fusion partner. A recombinant lysozyme was expressed in E. coli Rosetta cells using a pGEX-4T-1 expression vector, and the fusion protein was induced by ioporpyl-${\beta}$-D-thiogalactopyranoside (IPTG). The recombinant protein produced as an inclusion body was resolubilized in solubilization buffer, and the resultant solution was dialyzed in refolding buffer. After thrombin cleavage, the recombinant lysozyme was purified by ion exchange chromatography and reverse phase chromatography. The recombinant lysozyme was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis and immunoreactivity against the anti-Agrius lysozyme was observed by western blot analysis of this protein. The recombinant lysozyme displayed antibacterial activity against Bacillus megaterium and Micrococcus luteus, which was confirmed by the inhibition zone assay.

• Korean Journal of Microbiology
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• v.31 no.4
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• pp.274-278
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• 1993

Overproduced proteins in many cases result in forming insoluble inclusion bodies, and their formation might be due to high concentration of protein. To investigate how proteins become insoluble, chloramphenicol acetyltransferase (CAT) and .betha.-lactamase were overproduced, and their solubilities and activities were determined. CAT was accumulated from 9 to 45% of total cellular protein in a fully soluble form without inclusion body formation. CAT specific activity was shown to be proportional to the amount of the protein produced. Moderately produced .betha.-lactamase by the phase T7 expression system at 30.deg.C comprised only mature forms in a soluble form. However, overproduced .betha.-lactamase at 37.deg.C became insoluble. Most precursor forms of .betha.-lactamase in the cytoplasm were insoluble, whereas majority of the mature forms in the periplasm space were soluble. Also, chaperone GroE proteins which assist proper protein folding and translocation did not increase .betha.-lactamase solubility significantly under the experimental condition. It seems that the formation of inclusion bodies in the cell is related to the nature of protein itself rather than just to high concentration of protein.

• KSBB Journal
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• v.18 no.6
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• pp.500-505
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• 2003

‘LK (lipoprotein kringle) 68’is a polypeptide of a modified ansiostatin consisting of three kringle structures that might be clinically useful as a potential cancer therapeutics. It can be produced by overexpressing it as inclusion body in recombinant E. coli. In this study, solid-phase refolding processes using packed bed adsorption (PBA) and expanded bed adsorption (EBA) column were carried out to compare their refolding yields with that of the conventional, solution-phase refolding process, For the solution-phase and the PBA-mediated processes employing Q-Sepharose, washed inclusion body was used as the starting material, whereas both washed inclusion body and E. coli homogenate were used for the EBA-mediated process employing streamline DEAE. On the final recovery LK68 per unit mass of wet cell basis, the EBA- and PBA-mediated processes showed about 2.7- and 1.5-fold higher yields, respectively, than the solution-phase refolding method. The solid-phase refolded LK68 demonstrated the same Iysine binding bioactivity and the retention time in the RP-and SEC-HPLC as those of the native protein.