In vitro protein folding by E. coli ribosome: Unfolded protein splitting 70S to interact with 50S subunit

2008 ◽  
Vol 366 (2) ◽  
pp. 598-603 ◽  
Author(s):  
Arunima Basu ◽  
Dibyendu Samanta ◽  
Debasis Das ◽  
Saheli Chowdhury ◽  
Arpita Bhattacharya ◽  
...  
Keyword(s):  
Protein Folding ◽  
E Coli ◽  
Unfolded Protein ◽  
Vitro Protein

A PMMA microfluidic droplet platform for in vitro protein expression using crude E. coli S30 extract

Lab on a Chip ◽  
2009 ◽  
Vol 9 (23) ◽  
pp. 3391 ◽  
Author(s):  
N. Wu ◽  
Y. Zhu ◽  
S. Brown ◽  
J. Oakeshott ◽  
T. S. Peat ◽  
...  
Keyword(s):  
Protein Expression ◽  
E Coli ◽  
S30 Extract ◽  
In Vitro Protein Expression ◽  
Vitro Protein

In vitro protein synthesis directed by R17 viral ribonucleic acid. II. On the fate of mRNA in the cell-free system

1969 ◽  
Vol 47 (12) ◽  
pp. 1179-1186 ◽  
Author(s):  
Satomi J. Igarashi
Keyword(s):  
Amino Acids ◽  
Protein Synthesis ◽  
Amino Acid Incorporation ◽  
Free System ◽  
Cell Free System ◽  
E Coli ◽  
Acid Incorporation ◽  
Binding Factor ◽  
Vitro Protein

In the crude E. coli B cell-free system, mRNA was hydrolyzed by contaminating nuclease activities before significant polymerization of amino acids took place. Ribosomes appeared to be one of the sources of nuclease. A modified high-salt washing procedure was developed to remove nuclease from ribosomes. RNase-free ribosomes thus obtained appeared to be inactive in poly-U-directed phenylalanine incorporation, unless poly-U binding factor was added to the system. R17 RNA could not direct amino acid incorporation in the presence of RNase-free ribosomes because binding of intact R17 RNA to ribosomes did not take place even in the presence of poly-U binding factor.

scholarly journals Participation of X47-fluorescamine modified E. coli tRNAs in in vitro protein biosynthesis

1978 ◽  
Vol 5 (12) ◽  
pp. 4837-4854 ◽  
Author(s):  
Mathias Sprinzl ◽  
Heinz G. Faulhammer
Keyword(s):  
Protein Biosynthesis ◽  
E Coli ◽  
Vitro Protein

scholarly journals Understanding and exploiting interactions between cellular proteostasis pathways and infectious prion proteins for therapeutic benefit

Open Biology ◽  
2020 ◽  
Vol 10 (11) ◽  
pp. 200282
Author(s):  
Unekwu M. Yakubu ◽  
Celso S. G. Catumbela ◽  
Rodrigo Morales ◽  
Kevin A. Morano
Keyword(s):  
Cell Death ◽  
Protein Folding ◽  
Stress Response ◽  
Prion Proteins ◽  
Infectious Agent ◽  
Therapeutic Benefit ◽  
Model Systems ◽  
Unfolded Protein ◽  
Mouse Lines

Several neurodegenerative diseases of humans and animals are caused by the misfolded prion protein (PrP Sc ), a self-propagating protein infectious agent that aggregates into oligomeric, fibrillar structures and leads to cell death by incompletely understood mechanisms. Work in multiple biological model systems, from simple baker's yeast to transgenic mouse lines, as well as in vitro studies, has illuminated molecular and cellular modifiers of prion disease. In this review, we focus on intersections between PrP and the proteostasis network, including unfolded protein stress response pathways and roles played by the powerful regulators of protein folding known as protein chaperones. We close with analysis of promising therapeutic avenues for treatment enabled by these studies.

scholarly journals ERdj3, a Stress-inducible Endoplasmic Reticulum DnaJ Homologue, Serves as a CoFactor for BiP's Interactions with Unfolded Substrates

2005 ◽  
Vol 16 (1) ◽  
pp. 40-50 ◽  
Author(s):  
Ying Shen ◽  
Linda M. Hendershot
Keyword(s):  
Endoplasmic Reticulum ◽  
Protein Folding ◽  
Atpase Activity ◽  
Light Chain ◽  
Light Chains ◽  
Nonpermissive Temperature ◽  
Protein Substrates ◽  
Unfolded Protein

We recently identified ERdj3 as a component of unassembled immunoglobulin (Ig) heavy chain:BiP complexes. ERdj3 also associates with a number of other protein substrates, including unfolded light chains, a nonsecreted Ig light chain mutant, and the VSV-G ts045 mutant at the nonpermissive temperature. We produced an ERdj3 mutant that was unable to stimulate BiP's ATPase activity in vitro or to bind BiP in vivo. This mutant retained the ability to interact with unfolded protein substrates, suggesting that ERdj3 binds directly to proteins instead of via interactions with BiP. BiP remained bound to unfolded light chains longer than ERdj3, which interacted with unfolded light chains initially, but quickly disassociated before protein folding was completed. This suggests that ERdj3 may bind first to substrates and serve to inhibit protein aggregation until BiP joins the complex, whereas BiP remains bound until folding is complete. Moreover, our findings support a model where interactions with BiP help trigger the release of ERdj3 from the substrate:BiP complex.

scholarly journals FK506 Binding Protein from the Hyperthermophilic Archaeon Pyrococcus horikoshii Suppresses the Aggregation of Proteins in Escherichia coli

2002 ◽  
Vol 68 (2) ◽  
pp. 464-469 ◽  
Author(s):  
Akira Ideno ◽  
Masahiro Furutani ◽  
Yoshitaka Iba ◽  
Yoshikazu Kurosawa ◽  
Tadashi Maruyama
Keyword(s):  
Escherichia Coli ◽  
Protein Folding ◽  
Binding Protein ◽  
Fab Fragment ◽  
Ppiase Activity ◽  
Pyrococcus Horikoshii ◽  
E Coli ◽  
Fk506 Binding Protein ◽  
Soluble Recombinant Protein

ABSTRACT The 29-kDa FK506 binding protein (FKBP) gene is the only peptidyl-prolyl cis-trans isomerase (PPIase) gene in the genome of Pyrococcus horikoshii. We characterized the function of this FKBP (PhFKBP29) and used it to increase the production yield of soluble recombinant protein in Escherichia coli. The PPIase activity (k cat/Km ) of PhFKBP29 was found to be much lower than that of other archaeal 16- to 18-kDa FKBPs by a chymotrypsin-coupled assay of the oligo-peptidyl substrate at 15�C. Besides this low PPIase activity, PhFKBP29 showed chaperone-like protein folding activity which enhanced the refolding yield of chemically unfolded rhodanese in vitro. In addition, it suppressed thermal protein aggregation in a temperature range of 45 to 100�C. When the PhFKBP29 gene was coexpressed with the recombinant Fab fragment gene of the anti-hen egg lysozyme antibody in the cytoplasm of E. coli, whose expressed product tended to form an inactive aggregate in E. coli, it improved the yield of the soluble Fab fragments with antibody specificity. PhFKBP29 exerted protein folding and aggregation suppression in E. coli cells.

scholarly journals Conversion of a soluble protein into a potent chaperone in vivo

2018 ◽  
Author(s):  
Soon Bin Kwon ◽  
Kisun Ryu ◽  
Ahyun Son ◽  
Hotcherl Jeong ◽  
Keo-Heun Lim ◽  
...  
Keyword(s):  
Protein Folding ◽  
Soluble Protein ◽  
Protein Solubility ◽  
Chaperone Activity ◽  
Surface Charges ◽  
E Coli ◽  
Cellular Factors ◽  
Aggregation Inhibition

AbstractProtein-folding assistance and aggregation inhibition by cellular factors are largely understood in the context of molecular chaperones. As an alternative and complementary model, we previously proposed that, in general, soluble cellular macromolecules including chaperones with large excluded volume and surface charges exhibit the intrinsic chaperone activity to prevent aggregation of their connected polypeptides, irrespective of the connection types, and thus to aid productive protein folding. As a proof of concept, we here demonstrated that a model soluble protein with an inactive protease domain robustly exerted chaperone activity toward various proteins harboring a short protease-recognition tag of 7 residues in Escherichia coli. The chaperone activity of this protein was similar or even superior to that of representative E. coli chaperones in vivo. Furthermore, in vitro refolding experiments confirmed the in vivo results. Our findings revealed that a soluble protein exhibits the intrinsic chaperone activity, which is manifested, upon binding to aggregation-prone proteins. This study gives new insights into the ubiquitous chaperoning role of cellular macromolecules in protein-folding assistance and aggregation inhibition underlying the maintenance of protein solubility and proteostasis in vivo.

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