Protein refolding
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Human interleukin-3 (IL-3) is a hematopoietic growth factor involved in the survival, proliferation and differentiation of multipotent hematopoietic cells. However, recombinant IL-3 is usually expressed as insoluble form (inclusion bodies) in Escherichia coli cells.
8p vicaptainmarvel 21-04-2023 4 2 Download
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A high density lipoprotein (HDL)-linked enzyme with antioxidant and antiatherogenic properties, paraoxonase1(PON1), prevents the formation of atherosclerotic lesions in humans. In the present study, a recombinant hPON1 gene was produced using a small ubiquitin-related modifier (SUMO) fusion protein expression system. To that end, the hPON1 gene was amplified from human liver-ready cDNA, cloned into the expression vector pET SUMO, and expressed in Escherichia coli BL21 (DE3).
13p langthannam 29-12-2021 8 1 Download
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Undifferentiated pleomorphic sarcoma (UPS) is a heterogeneous tumor group, and little is known about molecular target therapy for UPS. Heat shock protein 90 (HSP90) is an expressed chaperone that refolds certain denatured proteins under stress conditions.
11p vimoscow2711 29-08-2020 9 2 Download
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The major wheat seed proteins are storage proteins that are synthesized in the rough endoplasmic reticulum (ER) of starchy endosperm cells. Many of these proteins have intra- and intermolecular disulfide bonds.
16p viminato2711 22-05-2020 16 1 Download
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Manganese peroxidase (MnP) from Irpex lacteus F17 has been shown to have a strong ability to degrade recalcitrant aromatic pollutants. In this study, a recombinant MnP from I. lacteus F17 was expressed in Escherichia coli Rosetta (DE3) in the form of inclusion bodies, which were refolded to achieve an active enzyme.
15p vihamax2711 21-04-2020 9 0 Download
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Proteins in inclusion bodies (IBs) present native-like secondary structures. However, chaotropic agents at denaturing concentrations, which are widely used for IB solubilization and subsequent refolding, unfold these secondary structures.
10p vihamax2711 21-04-2020 10 0 Download
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Virus-like particle (VLP) platform represents a promising approach for the generation of efficient and immunogenic subunit vaccines. Here, the feasibility of using grapevine fanleaf virus (GFLV) VLPs as a new carrier for the presentation of human papillomavirus (HPV) L2 epitope was studied.
12p vihamax2711 21-04-2020 13 0 Download
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The technology of recombinant single chain variable fragments (scFvs) expression has been used in research, diagnosis and treatment of diseases. In the previous study, we studied the expression of a recombinant single chain variable fragment recognizing blood A antigen (antiA-scFv) in E. coli. However, the protein was insoluble form resulting in difficulty for purification, refolding and activity assesment. Here, we present the study on fused expression of the recombinant scFv - specific blood A antigen with thioredoxin (Trx) in the expression vector pET32a(+).
8p gaunguyen6789 22-10-2019 10 1 Download
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Shikimate kinase was chosen as a convenient representative example of the subclass of a/b proteins with which to examine the mechanism of protein folding. In this paper we report on the refolding of the enzyme after denaturation in urea. As shown by the changes in secondary and tertiary structure monitored by far UV circular dichroism (CD) and fluorescence, respectively, the enzyme was fully unfolded in 4 M urea. From an analysis of the unfolding curve in terms of the two-state model, the stability of the folded state could be estimated as 17 kJÆmol)1.
9p system191 01-06-2013 43 3 Download
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Porcine heart cytoplasmic malate dehydrogenase (s-MDH) is a dimeric protein (2 · 35 kDa). We have studied equilibrium unfolding and refolding of s-MDH using activity assay, fluorescence, far-UV and near-UV circular dichroism (CD) spectroscopy, hydrophobic probe-1-anilino8-napthalene sulfonic acid binding, dynamic light scattering, and chromatographic (HPLC) techniques. The unfolding and refolding transitions are reversible and show the presence of two equilibrium intermediate states.
11p system191 01-06-2013 34 3 Download
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Heterologous expression of the extracellular domains (ECDs) of the nicotinic acetylcholine receptor (AChR) subunits may give large amounts of proteins for studying the functional and spatial characteristics of their ligand-binding sites. The ECD of the a7 subunit of the homo-oligomeric a7 neuronal AChR appears to be a more suitable object than the ECDs of other heteromeric neuronal or muscle-type AChRs. The rat a7 ECDs (amino-acid residues 1–210) were recently expressed in Escherichia coli as fusion proteins with maltose-binding protein...
9p system191 01-06-2013 35 2 Download
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Protein refolding is currently a fundamental problem in biophysics and molecular biology. We have studied the refolding process of frutalin, a tetrameric lectin that presents structural homology with jacalin but shows a more marked biological activity. The initial state in our refolding puzzle was that proteins were unfolded after thermal denaturation or denaturation induced by guanidine hydrochloride, and under both conditions, frutalin was refolded.
6p research12 01-06-2013 38 3 Download
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Each protein has a specific behavior and many parameters can vary (Stevens 2000). Protein'eXpert 1 has developed an expertise to determine optimal production conditions for recombinant proteins, and it provides several products and services in this field. One of these services is called the feasibility study which is a complete and standardized protein production study including cloning, expression and solubility tests, cell fractionation, purification, refolding assay (when necessary), quality control and delivery of 1-10 mg of soluble proteins. ...
4p bunthai_1 06-05-2013 41 2 Download
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Until now, only a small amount of information is available about tomato allergens. In the present study, a glycosylated allergen of tomato (Lycopersicon esculentum), Lyc e 2, was purified from tomato extract by a two-step FPLC method. The cDNA of two different isoforms of the protein, Lyc e 2.01 and Lyc e 2.02, was cloned into the bacterial expressionvector pET100D. The recombinant proteinswere purified by electroelution and refolded.
11p tumor12 20-04-2013 30 2 Download
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Staphylococcal nuclease (SNase) is a model protein that contains one domain and no disulfide bonds. Its stability in the native state may be maintained mainly by key amino acids. In this study, two point-mutated proteins each with a single base substitution [alanine for tryptophan (W140A) and alanine for lysine (K133A)] and two truncated fragment proteins {positions 1–139 [SNase(1–139) or W140O] and positions 1–141 [SNase(1–141) or E142O]} were generated.
7p fptmusic 11-04-2013 37 1 Download
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D-Ornithine aminomutase from Clostridium sticklandii comprises two strongly associating subunits, OraS and OraE, with molecular masses of 12 800 and 82 900 Da. Previous studies have shown that inEscherichia colithe recombinant OraS protein is synthesized in the soluble form and OraE as inclusion bodies. Refolding experiments also indicate that the interactions between OraS and OraE and the binding of either pyridoxal phosphate (PLP) or aden-osylcobalamin (AdoCbl) play important roles in the refolding process....
0p awards 05-04-2013 17 1 Download
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Superantigens (SAgs) are a class of disease-causing and immunostimulatory proteins of bacterial or viral origin that activate a large number of T-cells through interaction with the Vbdomain of T-cell receptors (TCRs). In this study, recombinant TCRbchains were constructed with human variable domains Vb5.2, Vb1and Vb2.1, expressed as inclusion bodies, refolded and purified. The Streptococcus pyogenesSAg SSA-1 was cloned and expressed as a soluble periplasmicprotein. SSA-1wasobtainedbothas amonomer and a dimer that has an intermolecular disulfide bond. ...
9p awards 05-04-2013 35 3 Download
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The Mycobacterium tuberculosisgenome contains an unusually high number of proteins involved in the metabo-lism of lipids belonging to the Lip family, including various nonlipolytic and lipolytic hydrolases. Driven by a structural genomic approach, we have biochemically characterized the Rv1399c gene product, LipH, previously annotated as a putative lipase. Rv1399c was overexpressed inE. colias inclusion bodies and refolded. Rv1399c efficiently hydro-lyzes soluble triacylglycerols and vinyl esters.
0p awards 05-04-2013 46 1 Download
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Cyclophilins facilitate the peptidyl-prolyl isomerization of atrans-isomer to a cis-isomer in the refolding process of unfolded proteins to recover the natural folding state with cis-proline conformation.Todate, only short peptideswitha cis-formproline have been observed in complexes of human and Escherichia coliproteins of cyclophilin A, which is present in cytoplasm. The crystal structures analyzed in this study show two complexes inwhich peptides having atrans-form proline, i.e.
10p awards 05-04-2013 26 2 Download
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Searching for bioactive peptides, we analyzed acidic extracts ofPhyllomedusa sauvagiiskin and found two new proteins, PSKP-1 and PSKP-2, of 6.7 and 6.6 kDa, respectively, which, by sequence homology, belong to the Kazal family of serine protease inhibitors. PSKP-1 and PSKP-2 exhibit the unprecedented feature of having proline at P1and P2 positions. A gene encoding PSKP-1 was synthesized and expressed in Escherichia coli. Recombinant PSKP-1 was purified from inclusion bodies, oxidatively refolded to the native state, and characterized by chemical, hydrodynamic andoptical studies. ...
10p dell39 03-04-2013 38 3 Download