scholarly journals Protein Renaturation

2020 ◽  
Author(s):  
Keyword(s):  
Protein Renaturation

Optimizing a production strategy for a nonspecific nuclease from Yersinia enterocolitica subsp. palearctica in genetically engineered Escherichia coli

2019 ◽  
Vol 366 (24) ◽  
Author(s):  
Yan Ge ◽  
Senlin Guo ◽  
Tao Liu ◽  
Chen Zhao ◽  
Duanhua Li ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Yersinia Enterocolitica ◽  
Inclusion Bodies ◽  
Genetically Engineered ◽  
Biological Research ◽  
Dilution Method ◽  
E Coli ◽  
Protein Renaturation ◽  
Engineered Escherichia Coli ◽  
Pharmaceutical Production

ABSTRACT A nuclease from Yersinia enterocolitica subsp. palearctica (Nucyep) is a newly found thermostable nonspecific nuclease. The heat-resisting ability of this nuclease would be extremely useful in biological research or pharmaceutical production. However, the application of this nuclease is limited because of its poor yield. This research aimed to improve Nucyep productivity by producing a novel genetically engineered Escherichia coli and optimizing the production procedures. After 4 h of induction by lactose, the new genetically engineered E. coli can express a substantial amount of Nucyep in the form of inclusion bodies. The yield was approximately 0.3 g of inclusion bodies in 1 g of bacterial pellets. The inclusion bodies were extracted by sonication and solubilized in an 8 M urea buffer. Protein renaturation was successfully achieved by dilution method. Pure enzyme was obtained after subjecting the protein solution to anion exchange. The Nucyep showed its nonspecific and heat resistant properties as previously reported (Boissinot et  al. 2016). Through a quantification method, its activity was determined to be 1.3 × 10 6 Kunitz units (K.U.)/mg. These results can serve as a reference for increasing Nucyep production.

scholarly journals Protein renaturation by the liquid organic salt ethylammonium nitrate

2000 ◽  
Vol 9 (10) ◽  
pp. 2001-2008 ◽  
Author(s):  
Catherine A. Summers ◽  
Robert A. Flowers
Keyword(s):  
Organic Salt ◽  
Protein Renaturation ◽  
Ethylammonium Nitrate

A Sparse Matrix Screen to Establish Initial Conditions for Protein Renaturation

1995 ◽  
Vol 230 (1) ◽  
pp. 8-15 ◽  
Author(s):  
A. Hofmann ◽  
M. Tai ◽  
W. Wong ◽  
C.G. Glabe
Keyword(s):  
Initial Conditions ◽  
Sparse Matrix ◽  
Protein Renaturation

scholarly journals Non-detergent sulphobetaines: a new class of molecules that facilitate in vitro protein renaturation

1996 ◽  
Vol 1 (1) ◽  
pp. 21-27 ◽  
Author(s):  
Michel E Goldberg ◽  
Nicole Expert-Bezançon ◽  
Laurent Vuillard ◽  
Thierry Rabilloud
Keyword(s):  
New Class ◽  
Protein Renaturation ◽  
Vitro Protein

scholarly journals Chaperone properties of Escherichia coli thioredoxin and thioredoxin reductase

2003 ◽  
Vol 371 (3) ◽  
pp. 965-972 ◽  
Author(s):  
Renée KERN ◽  
Abderrahim MALKI ◽  
Arne HOLMGREN ◽  
Gilbert RICHARME
Keyword(s):  
Escherichia Coli ◽  
Citrate Synthase ◽  
Thioredoxin Reductase ◽  
Cellular Protein ◽  
Unfolded Proteins ◽  
Urea Denaturation ◽  
Isomerase Activity ◽  
Protein Renaturation ◽  
Thioredoxin System ◽  
Pancreatic Trypsin Inhibitor

Thioredoxin, thioredoxin reductase and NADPH form the thioredoxin system and are the major cellular protein disulphide reductase. We report here that Escherichia coli thioredoxin and thioredoxin reductase interact with unfolded and denatured proteins, in a manner similar to that of molecular chaperones that are involved in protein folding and protein renaturation after stress. Thioredoxin and/or thioredoxin reductase promote the functional folding of citrate synthase and α-glucosidase after urea denaturation. They also promote the functional folding of the bacterial galactose receptor, a protein without any cysteines. Furthermore, redox cycling of thioredoxin/thioredoxin reductase in the presence of NADPH and cystine stimulates the renaturation of the galactose receptor, suggesting that the thioredoxin system functions like a redox-powered chaperone machine. Thioredoxin reductase prevents the aggregation of citrate synthase under heat-shock conditions. It forms complexes that are more stable than those formed by thioredoxin with several unfolded proteins such as reduced carboxymethyl α-lactalbumin and unfolded bovine pancreatic trypsin inhibitor. These results suggest that the thioredoxin system, in addition to its protein disulphide isomerase activity possesses chaperone-like properties, and that its thioredoxin reductase component plays a major role in this function.

High throughput systems and mathematical models for prediction of protein renaturation processes

2014 ◽  
Vol 31 ◽  
pp. S126-S127
Author(s):  
Bernhard Mißbichler ◽  
Cornelia Walther ◽  
Sabrina Mayer ◽  
Dorota Antos ◽  
Alois Jungbauer ◽  
...  
Keyword(s):  
Mathematical Models ◽  
High Throughput ◽  
Protein Renaturation

scholarly journals Characterization of an Aminoacylase from the Hyperthermophilic Archaeon Pyrococcus furiosus

2001 ◽  
Vol 183 (14) ◽  
pp. 4259-4268 ◽  
Author(s):  
Sherry V. Story ◽  
Amy M. Grunden ◽  
Michael W. W. Adams
Keyword(s):  
Amino Acids ◽  
Recombinant Protein ◽  
Pyrococcus Furiosus ◽  
Protein A ◽  
Genome Database ◽  
Hyperthermophilic Archaeon ◽  
Protein Renaturation ◽  
Cell Extracts ◽  
Growth Substrates ◽  
Catalytically Active

ABSTRACT Aminoacylase was identified in cell extracts of the hyperthermophilic archaeon Pyrococcus furiosus by its ability to hydrolyze N-acetyl-l-methionine and was purified by multistep chromatography. The enzyme is a homotetramer (42.06 kDa per subunit) and, as purified, contains 1.0 ± 0.48 g-atoms of zinc per subunit. Treatment of the purified enzyme with EDTA resulted in complete loss of activity. This was restored to 86% of the original value (200 U/mg) by treatment with ZnCl2 (and to 74% by the addition of CoCl2). After reconstitution with ZnCl2, the enzyme contained 2.85 ± 0.48 g-atoms of zinc per subunit. Aminoacylase showed broad substrate specificity and hydrolyzed nonpolarN-acylated l amino acids (Met, Ala, Val, and Leu), as well as N-formyl-l-methionine. The high Km values for these compounds indicate that the enzyme plays a role in the metabolism of protein growth substrates rather than in the degradation of cellular proteins. Maximal aminoacylase activity withN-acetyl-l-methionine as the substrate occurred at pH 6.5 and a temperature of 100°C. The N-terminal amino acid sequence of the purified aminoacylase was used to identify, in theP. furiosus genome database, a gene that encodes 383 amino acids. The gene was cloned and expressed in Escherichia coli by using two approaches. One involved the T7lac promoter system, in which the recombinant protein was expressed as inclusion bodies. The second approach used the Trx fusion system, and this produced soluble but inactive recombinant protein. Renaturation and reconstitution experiments with Zn2+ ions failed to produce catalytically active protein. A survey of databases showed that, in general, organisms that contain a homolog of theP. furiosus aminoacylase (≥50% sequence identity) utilize peptide growth substrates, whereas those that do not contain the enzyme are not known to be proteolytic, suggesting a role for the enzyme in primary catabolism.

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