In Vitro Refolding of Cyclomaltodextrin Glucanotransferase from Cytoplasmic Inclusion Bodies Formed upon Expression in Escherichia coli

1995 ◽  
Vol 6 (1) ◽  
pp. 56-62 ◽  
Author(s):  
J. Hellman ◽  
P. Lassila ◽  
P. Mantsala
Keyword(s):  
Escherichia Coli ◽  
Inclusion Bodies ◽  
Cytoplasmic Inclusion ◽  
Expression In Escherichia Coli ◽  
Cytoplasmic Inclusion Bodies ◽  
Cyclomaltodextrin Glucanotransferase ◽  
In Vitro Refolding

Abstract 4: Macrophage p62/SQSTM1 Ameliorates Atherosclerosis by Sequestering Inclusion Bodies and Mediating Mitophagy

2015 ◽  
Vol 35 (suppl_1) ◽  
Author(s):  
Ismail Sergin ◽  
Somashubhra Bhattacharya ◽  
Carl J Stokes ◽  
John A Curci ◽  
Babak Razani
Keyword(s):  
Inclusion Bodies ◽  
Cytoplasmic Inclusion ◽  
Protein Aggregates ◽  
Whole Body ◽  
Plaque Burden ◽  
Protein Accumulation ◽  
Inflammasome Activation ◽  
Cytoplasmic Inclusions ◽  
Cytoplasmic Inclusion Bodies

Protein and organelle turnover is critical for cellular homeostasis and is prominently mediated by autophagy. Disruptions in autophagy lead to accumulation of protein aggregates and dysfunctional organelles such as mitochondria. Recent evidence suggests that the chaperone protein p62 is a critical link for targeting polyubiquitinated protein aggregates/damaged mitochondria to autophagosomes for degradation. Herein we describe a p62-centric mechanism of handling protein aggregates and dysfunctional mitochondria in atherosclerosis. Macrophages deficient in autophagy (ATG5-/-) or rendered deficient by incubation with atherogenic lipids have significantly increased levels of p62. This coincides with 1) the accumulation of polyubiquitinated proteins co-localizing with p62 and present as cytoplasmic inclusion bodies, and 2) p62 co-localization with mitochondrial markers. Aortas from atherosclerotic (ApoE-/-) mice also have progressive and marked elevations in p62, polyubiquitinated proteins, and mitochondrial reactive oxygen species that predominantly co-localize with plaque macrophages, a process further exacerbated in the autophagy-deficient setting. The formation of cytoplasmic inclusions and maintenance of adequate mitochondrial function appears to be dependent on p62. Lipid-loaded p62-null macrophages show polyubiquitinated protein accumulation present in a diffuse/disrupted cytoplasmic pattern. These macrophages also develop larger dysmorphic mitochondria with increased polarization and decreased oxidative phosphorylation capacity. As a result, p62-null macrophages display apoptotic susceptibility to atherogenic lipids and increased IL-1β secretion likely through mitochondrial-dependent inflammasome activation. Consistent with our in vitro observations, mice with either whole-body p62-deficiency or transplanted with p62-deficient bone marrow show significantly increased atherosclerotic plaque burden and lesion complexity with increased apoptosis and necrotic cores. Taken together, these data demonstrate a previously unrecognized atheroprotective role for macrophage p62 by facilitating the formation of inclusion bodies and maintaining healthy mitochondria.

scholarly journals Minimal Elements Required for the Formation of Respiratory Syncytial Virus Cytoplasmic Inclusion Bodies In Vivo and In Vitro

mBio ◽  
2020 ◽  
Vol 11 (5) ◽  
Author(s):  
Marie Galloux ◽  
Jennifer Risso-Ballester ◽  
Charles-Adrien Richard ◽  
Jenna Fix ◽  
Marie-Anne Rameix-Welti ◽  
...  
Keyword(s):  
Respiratory Syncytial Virus ◽  
Liquid Phase ◽  
Inclusion Bodies ◽  
Cytoplasmic Inclusion ◽  
Cytoplasmic Inclusion Bodies ◽  
Syncytial Virus ◽  
P Proteins ◽  
Nucleoprotein N ◽  
Liquid Liquid Phase Separation

ABSTRACT Infection of host cells by the respiratory syncytial virus (RSV) is characterized by the formation of spherical cytoplasmic inclusion bodies (IBs). These structures, which concentrate all the proteins of the polymerase complex as well as some cellular proteins, were initially considered aggresomes formed by viral dead-end products. However, recent studies revealed that IBs are viral factories where viral RNA synthesis, i.e., replication and transcription, occurs. The analysis of IBs by electron microscopy revealed that they are membrane-less structures, and accumulated data on their structure, organization, and kinetics of formation revealed that IBs share the characteristics of cellular organelles, such as P-bodies or stress granules, suggesting that their morphogenesis depends on a liquid-liquid phase separation mechanism. It was previously shown that expression of the RSV nucleoprotein N and phosphoprotein P of the polymerase complex is sufficient to induce the formation of pseudo-IBs. Here, using a series of truncated P proteins, we identified the domains of P required for IB formation and show that the oligomeric state of N, provided it can interact with RNA, is critical for their morphogenesis. We also show that pseudo-IBs can form in vitro when recombinant N and P proteins are mixed. Finally, using fluorescence recovery after photobleaching approaches, we reveal that in cellula and in vitro IBs are liquid organelles. Our results strongly support the liquid-liquid phase separation nature of IBs and pave the way for further characterization of their dynamics. IMPORTANCE Respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract illness in infants, elderly, and immunocompromised people. No vaccine or efficient antiviral treatment is available against this virus. The replication and transcription steps of the viral genome are appealing mechanisms to target for the development of new antiviral strategies. These activities take place within cytoplasmic inclusion bodies (IBs) that assemble during infection. Although expression of both the viral nucleoprotein (N) and phosphoprotein (P) allows induction of the formation of these IBs, the mechanism sustaining their assembly remains poorly characterized. Here, we identified key elements of N and P required for the scaffolding of IBs and managed for the first time to reconstitute RSV pseudo-IBs in vitro by coincubating recombinant N and P proteins. Our results provide strong evidence that the biogenesis of RSV IBs occurs through liquid-liquid phase transition mediated by N-P interactions.

scholarly journals Chemical Assistance in Refolding of Bacterial Inclusion Bodies

2011 ◽  
Vol 2011 ◽  
pp. 1-6 ◽  
Author(s):  
Mona Alibolandi ◽  
Hasan Mirzahoseini
Keyword(s):  
Escherichia Coli ◽  
Recombinant Proteins ◽  
Inclusion Bodies ◽  
Industrial Processes ◽  
Small Scale ◽  
Heterologous Proteins ◽  
E Coli ◽  
Major Bottleneck ◽  
In Vitro Refolding

Escherichia coliis one of the most widely used hosts for the production of recombinant proteins but insoluble expression of heterologous proteins is a major bottleneck in production of recombinant proteins inE. coli.In vitrorefolding of inclusion body into proteins with native conformations is a solution for this problem but there is a need for optimization of condition for each protein specifically. Several approaches have been described for in vitro refolding; most of them involve the use of additives for assisting correct folding. Cosolutes play a major role in refolding process and can be classified according to their function as aggregation suppressors and folding enhancers. This paper presents a review of additives that are used in refolding process of insoluble recombinant proteins in small scale and industrial processes.

scholarly journals Apo(a)-kringle IV-type 6: expression in Escherichia coli, purification and in vitro refolding

2000 ◽  
Vol 13 (9) ◽  
pp. 661-666 ◽  
Author(s):  
A. Hrzenjak ◽  
S. Frank ◽  
B. Maderegger ◽  
H. Sterk ◽  
G.M. Kostner
Keyword(s):  
Escherichia Coli ◽  
Expression In Escherichia Coli ◽  
In Vitro Refolding
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