• Journal of Microbiology and Biotechnology
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• 제6권4호
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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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• 제16권1호
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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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• 제6권1호
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• pp.111-114
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• 1991

대장균의 plasmid상의 ${\beta}-lactamase$를 IPTG induction으로 대량 생산할 때 precursor processing inhibitor (CCCP)를 가하여 ${\beta}-lactamase$의 soluble fraction과 insoluble fraction (inclusion body)의 생산성을 비교하였다. CCCP로 처리한 경우가 더 많은 soluble ${\beta}-lactamase$를 생성하였으나,inclusion body의 양에는 큰 차이가 없었다. 이것은 ${\beta}-lactamase$ precursor processing 속도를 낮출 경우 soluble ${\beta}-lactamase$가 더 많이 생성된다는 것을 보여주었다.

• 대한수의학회지
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• 제22권1호
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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.

• 대한수의학회지
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• 제21권1호
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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.

• 한국수산과학회지
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• 제44권5호
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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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• 제30권2호
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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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• 제16권6호
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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.

• 미생물학회지
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• 제31권4호
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• pp.274-278
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• 1993

단백질이 어떻게 비수용성이 되는지를 알기위해, 클로람페니콜 아세틸전이효소와 베타-락타메이즈를 과잉생산하여 그들의 수용성과 활성을 측정하였다. 클로람페니콜 아세틸전이효소는 총단백질의 9에서 45%를 차지하였으며, inclusion body 형성없이 완전히 수용성이었으며, 효소활성은 만들어진 양과 비례하였다. 또한 30℃에서 T7 발현체계에 의해 생성된 베타-락타메이즈는 수용성의 숙성체였으나, 37℃에서는 비수용성이 되었다. 세포질에 있는 대부분의 베타-락타메이즈는 비수용성이었고. 페리플라즘 공간에서는 대부분이 수용성이었다. 단백질의 올바른 폴딩을 도와주는 chaperone의 일종인 GroEL 단백질은 본 실험조선에서는 베타-락타베이즈의 수용성을 별로 높이지는 못했다. 세포 내에서 inclusion body의 형성은 단백질의 높은 종도보다는 각각 단밸질 자체의 특성과 관련된 듯하다.

• KSBB Journal
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• 제18권6호
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• pp.500-505
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• 2003

재조합 대장균에서 내포체 형태로 발현시킨 LK68을 생물학적 활성을 가진 native한 단백질로 재생시키기 위해서 PBA 크로마토그래피와 EBA 크로마토그래피를 이용한 고체상 재접힘을 수행하였다. 내포체와 세포파쇄액을 시작물질로 하여 재접힘 공정을 수행하였으며 총 단백질 회수율과 재접힘 수율을 비교한 결과, EBA 공정이 기존의 액상 재접힘이나 PBA를 이용한 재접힘 공정에 비하여 우수함을 확인하였다. 또한 Iysine binding, RP-HPLC, SEC-HPLC, Ellman method 등을 사용하여 분석한 결과 재접힘된 LK68이 native LK68와 동등함을 확인하였다. 본 연구를 통해 EBA 크로마토그래피를 이용한 재접힘 방법은 재접힘 단계의 수율을 향상시킬 뿐 아니라 공정 단계, 시간 등을 감소시켜 공정 성능을 전체적으로 향상시킬 수 있음을 제시하였다.