Protein expression and refolding – A practical guide to getting the most out of inclusion bodies

2004 ◽  
pp. 31-50 ◽  
Author(s):  
Lisa D. Cabrita ◽  
Stephen P. Bottomley
Keyword(s):  
Protein Expression ◽  
Inclusion Bodies ◽  
Practical Guide

scholarly journals Function of the N-terminal propeptide of an aminopeptidase from Vibrio proteolyticus

2000 ◽  
Vol 350 (3) ◽  
pp. 671-676 ◽  
Author(s):  
Zhen-Zhong ZHANG ◽  
Satoru NIRASAWA ◽  
Yoshiaki NAKAJIMA ◽  
Michiteru YOSHIDA ◽  
Kiyoshi HAYASHI
Keyword(s):  
Escherichia Coli ◽  
Enzyme Activity ◽  
Active Region ◽  
Protein Expression ◽  
Inclusion Bodies ◽  
Active Enzyme ◽  
Vibrio Proteolyticus ◽  
Show Evidence ◽  
Inhibited Enzyme

An aminopeptidase from Vibrio proteolyticus was translated as a preproprotein consisting of four domains: a signal peptide, an N-terminal propeptide, a mature region and a C-terminal propeptide. Protein expression and analysis of the activity results demonstrated that the N-terminal propeptide was essential to the formation of the active enzyme in Escherichia coli. Urea dissolution of inclusion bodies and dialysis indicated that the N-terminal propeptide could facilitate the correct folding of the enzyme in vitro. Using l-Leu-p-nitroanilide as the substrate, the kinetic parameters (kcat and Km) of the pro-aminopeptidase and processed aminopeptidases were analysed. The results suggested that the N-terminal propeptide inhibited enzyme activity of the mature region. In contrast, the C-terminal propeptide did not show evidence of forming an active enzyme, of correctly folding in vitro or of inhibiting the active region.

Cytosolic Ca2+ regulates protein expression in E. coli through release from inclusion bodies

2007 ◽  
Vol 360 (1) ◽  
pp. 33-39 ◽  
Author(s):  
Riffat Naseem ◽  
Sally Rosser Davies ◽  
Helen Jones ◽  
Kenneth T. Wann ◽  
I. Barry Holland ◽  
...  
Keyword(s):  
Protein Expression ◽  
Inclusion Bodies ◽  
E Coli

scholarly journals A novel protein fusion partner, carbohydrate-binding module family 66, to enhance heterologous protein expression in Escherichia coli

2021 ◽  
Vol 20 (1) ◽  
Author(s):  
Hyunjun Ko ◽  
Minsik Kang ◽  
Mi-Jin Kim ◽  
Jiyeon Yi ◽  
Jin Kang ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Protein Expression ◽  
Inclusion Bodies ◽  
Heterologous Protein ◽  
Soluble Expression ◽  
Carbohydrate Binding ◽  
Heterologous Protein Expression ◽  
Carbohydrate Binding Module ◽  
Fusion Tag ◽  
E Coli

Abstract Background Proteins with novel functions or advanced activities developed by various protein engineering techniques must have sufficient solubility to retain their bioactivity. However, inactive protein aggregates are frequently produced during heterologous protein expression in Escherichia coli. To prevent the formation of inclusion bodies, fusion tag technology has been commonly employed, owing to its good performance in soluble expression of target proteins, ease of application, and purification feasibility. Thus, researchers have continuously developed novel fusion tags to expand the expression capacity of high-value proteins in E. coli. Results A novel fusion tag comprising carbohydrate-binding module 66 (CBM66) was developed for the soluble expression of heterologous proteins in E. coli. The target protein solubilization capacity of the CBM66 tag was verified using seven proteins that are poorly expressed or form inclusion bodies in E. coli: four human-derived signaling polypeptides and three microbial enzymes. Compared to native proteins, CBM66-fused proteins exhibited improved solubility and high production titer. The protein-solubilizing effect of the CBM66 tag was compared with that of two commercial tags, maltose-binding protein and glutathione-S-transferase, using poly(ethylene terephthalate) hydrolase (PETase) as a model protein; CBM66 fusion resulted in a 3.7-fold higher expression amount of soluble PETase (approximately 370 mg/L) compared to fusion with the other commercial tags. The intact PETase was purified from the fusion protein upon serial treatment with enterokinase and affinity chromatography using levan-agarose resin. The bioactivity of the three proteins assessed was maintained even when the CBM66 tag was fused. Conclusions The use of the CBM66 tag to improve soluble protein expression facilitates the easy and economic production of high-value proteins in E. coli.

scholarly journals The Ebola Virus Nucleoprotein Recruits the Nuclear RNA Export Factor NXF1 into Inclusion Bodies to Facilitate Viral Protein Expression

Cells ◽  
2020 ◽  
Vol 9 (1) ◽  
pp. 187 ◽  
Author(s):  
Lisa Wendt ◽  
Janine Brandt ◽  
Bianca S. Bodmer ◽  
Sven Reiche ◽  
Marie Luisa Schmidt ◽  
...  
Keyword(s):  
Life Cycle ◽  
Protein Expression ◽  
Viral Protein ◽  
Inclusion Bodies ◽  
Ebola Virus ◽  
Rna Binding ◽  
Nuclear Rna ◽  
Rna Export ◽  
Nuclear Rna Export

Ebola virus (EBOV) causes severe outbreaks of viral hemorrhagic fever in humans. While virus-host interactions are promising targets for antivirals, there is only limited knowledge regarding the interactions of EBOV with cellular host factors. Recently, we performed a genome-wide siRNA screen that identified the nuclear RNA export factor 1 (NXF1) as an important host factor for the EBOV life cycle. NXF1 is a major component of the nuclear mRNA export pathway that is usurped by many viruses whose life cycles include nuclear stages. However, the role of NXF1 in the life cycle of EBOV, a virus replicating in cytoplasmic inclusion bodies, remains unknown. In order to better understand the role of NXF1 in the EBOV life cycle, we performed a combination of co-immunoprecipitation and double immunofluorescence assays to characterize the interactions of NXF1 with viral proteins and RNAs. Additionally, using siRNA-mediated knockdown of NXF1 together with functional assays, we analyzed the role of NXF1 in individual aspects of the virus life cycle. With this approach we identified the EBOV nucleoprotein (NP) as a viral interaction partner of NXF1. Further studies revealed that NP interacts with the RNA-binding domain of NXF1 and competes with RNA for this interaction. Co-localization studies showed that RNA binding-deficient, but not wildtype NXF1, accumulates in NP-derived inclusion bodies, and knockdown experiments demonstrated that NXF1 is necessary for viral protein expression, but not for viral RNA synthesis. Finally, our results showed that NXF1 interacts with viral mRNAs, but not with viral genomic RNAs. Based on these results we suggest a model whereby NXF1 is recruited into inclusion bodies to promote the export of viral mRNA:NXF1 complexes from these sites. This would represent a novel function for NXF1 in the life cycle of cytoplasmically replicating viruses, and may provide a basis for new therapeutic approaches against EBOV, and possibly other emerging viruses.

Inclusion bodies in measles encephalitis

JAMA ◽  
1966 ◽  
Vol 195 (4) ◽  
pp. 307-308
Author(s):  
C. A. Phillips
Keyword(s):  
Inclusion Bodies

Origin of Hepatic Nuclear Inclusion Bodies

1973 ◽  
Vol 31 ◽  
pp. 426-427
Author(s):  
F. G. Zaki ◽  
J. A. Greenlee ◽  
C. H. Keysser
Keyword(s):  
Inclusion Bodies ◽  
Storage Disease ◽  
Glycogen Storage ◽  
Nutritional Deficiencies ◽  
Nuclear Inclusion ◽  
Electron Microscopic ◽  
Human Liver Cells ◽  
Microscopic Studies ◽  
Per Se ◽  
Experimental Liver

Nuclear inclusion bodies seen in human liver cells may appear in light microscopy as deposits of fat or glycogen resulting from various diseases such as diabetes, hepatitis, cholestasis or glycogen storage disease. These deposits have been also encountered in experimental liver injury and in our animals subjected to nutritional deficiencies, drug intoxication and hepatocarcinogens. Sometimes these deposits fail to demonstrate the presence of fat or glycogen and show PAS negative reaction. Such deposits are considered as viral products.Electron microscopic studies of these nuclei revealed that such inclusion bodies were not products of the nucleus per se but were mere segments of endoplasmic reticulum trapped inside invaginating nuclei (Fig. 1-3).

Electron Microscopy of a Herpesvirus Isolated from Marek's Disease in Duck and Chicken Embryo Fibroblast Cultures

1968 ◽  
Vol 26 ◽  
pp. 222-223 ◽  
Author(s):  
Keyvan Nazerian
Keyword(s):  
Inclusion Bodies ◽  
Chicken Embryo Fibroblast ◽  
Marek's Disease ◽  
Marek’S Disease ◽  
Duck Embryo Fibroblast ◽  
Multinucleated Cells ◽  
Viral Particles ◽  
Infected Cells ◽  
And Control ◽  
Do So

A herpes-like virus has been isolated from duck embryo fibroblast (DEF) cultures inoculated with blood from Marek's disease (MD) infected birds. Cultures which contained this virus produced MD in susceptible chickens while virus negative cultures and control cultures failed to do so. This and other circumstantial evidence including similarities in properties of the virus and the MD agent implicate this virus in the etiology of MD.Histochemical studies demonstrated the presence of DNA-staining intranuclear inclusion bodies in polykarocytes in infected cultures. Distinct nucleo-plasmic aggregates were also seen in sections of similar multinucleated cells examined with the electron microscope. These aggregates are probably the same as the inclusion bodies seen with the light microscope. Naked viral particles were observed in the nucleus of infected cells within or on the edges of the nucleoplasmic aggregates. These particles measured 95-100mμ, in diameter and rarely escaped into the cytoplasm or nuclear vesicles by budding through the nuclear membrane (Fig. 1). The enveloped particles (Fig. 2) formed in this manner measured 150-170mμ in diameter and always had a densely stained nucleoid. The virus in supernatant fluids consisted of naked capsids with 162 hollow, cylindrical capsomeres (Fig. 3). Enveloped particles were not seen in such preparations.

Studies of a Defective Measles Virus

1968 ◽  
Vol 26 ◽  
pp. 208-209
Author(s):  
R. M. McCombs ◽  
M. Benyesh-Melnick ◽  
J. P. Brunschwig
Keyword(s):  
Inclusion Bodies ◽  
Measles Virus ◽  
Giant Cells ◽  
Helical Structures ◽  
Thin Sections ◽  
Virus Particles ◽  
Extracellular Virus ◽  
Culture Cells ◽  
Infected Cells ◽  
Eosinophilic Inclusions

Measles virus is an agent that is capable of replicating in a number of different culture cells and generally causes the formation of multinucleated giant cells. As a result of infection, virus is released from the cells into the culture fluids and reinfection can be initiated by this cell-free virus. The extracellular virus has been examined by negative staining with phosphotungstic acid and has been shown to be a rather pleomorphic particle with a diameter of about 140 mμ. However, no such virus particles have been detected in thin sections of the infected cells. Rather, the only virus-induced structures present in the giant cells are eosinophilic inclusions (intracytoplasmic or intranuclear). These inclusion bodies have been shown to contain helical structures, resembling the nucleocapsid observed in negatively stained preparations.

Microprobe analysis of inclusion bodies related to two benzofuran derivatives

1987 ◽  
Vol 45 ◽  
pp. 672-673
Author(s):  
T. L. Benning ◽  
P. Ingram ◽  
J. D. Shelburne
Keyword(s):  
Inclusion Bodies ◽  
Uranyl Acetate ◽  
Multiple Organ ◽  
High Dose ◽  
En Bloc ◽  
Tissue Samples ◽  
Transmission Electron ◽  
Organ Systems ◽  
Dense Inclusion ◽  
Melanin Complex

Two benzofuran derivatives, chlorpromazine and amiodarone, are known to produce inclusion bodies in human tissues. Prolonged high dose chlorpromazine therapy causes hyperpigmentation of the skin with electron-dense inclusion bodies present in dermal histiocytes and endothelial cells ultrastructurally. The nature of the deposits is not known although a drug-melanin complex has been hypothesized. Amiodarone may also cause cutaneous hyperpigmentation and lamellar lysosomal inclusion bodies have been demonstrated within the cells of multiple organ systems. These lamellar bodies are believed to be the product of an amiodarone-induced phospholipid storage disorder. We performed transmission electron microscopy (TEM) and energy dispersive x-ray microanalysis (EDXA) on tissue samples from patients treated with these drugs, attempting to detect the sulfur atom of chlorpromazine and the iodine atom of amiodarone within their respective inclusion bodies.A skin biopsy from a patient with hyperpigmentation due to prolonged chlorpromazine therapy was fixed in 4% glutaraldehyde and processed without osmium tetroxide or en bloc uranyl acetate for Epon embedding.

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