Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Thermostable exoshells fold and stabilize recombinant proteins.

ABSTRACT: The expression and stabilization of recombinant proteins is fundamental to basic and applied biology. Here we have engineered a thermostable protein nanoparticle (tES) to improve both expression and stabilization of recombinant proteins using this technology. tES provides steric accommodation and charge complementation to green fluorescent protein (GFPuv), horseradish peroxidase (HRPc), and Renilla luciferase (rLuc), improving the yields of functional in vitro folding by ~100-fold. Encapsulated enzymes retain the ability to metabolize small-molecule substrates, presumably via four 4.5-nm pores present in the tES shell. GFPuv exhibits no spectral shifts in fluorescence compared to a nonencapsulated control. Thermolabile proteins internalized by tES are resistant to thermal, organic, chaotropic, and proteolytic denaturation and can be released from the tES assembly with mild pH titration followed by proteolysis.

SUBMITTER: Deshpande S

PROVIDER: S-EPMC5682286 | biostudies-literature | 2017 Nov

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Publications

Thermostable exoshells fold and stabilize recombinant proteins.

Deshpande Siddharth S Masurkar Nihar D ND Girish Vallerinteavide Mavelli VM Desai Malan M Chakraborty Goutam G Chan Juliana M JM Drum Chester L CL

Nature communications 20171113 1

The expression and stabilization of recombinant proteins is fundamental to basic and applied biology. Here we have engineered a thermostable protein nanoparticle (tES) to improve both expression and stabilization of recombinant proteins using this technology. tES provides steric accommodation and charge complementation to green fluorescent protein (GFPuv), horseradish peroxidase (HRPc), and Renilla luciferase (rLuc), improving the yields of functional in vitro folding by ~100-fold. Encapsulated ...[more]

PMID: 29129910

Similar Datasets

Fanconi anemia proteins stabilize replication forks.

Project description:Fanconi anemia (FA) is a recessive genetic disorder characterized by hypersensitivity to crosslinking agents that has been attributed to defects in DNA repair and/or replication. FANCD2 and the FA core complex bind to chromatin during DNA replication; however, the role of FA proteins during replication is unknown. Using Xenopus cell-free extracts, we show that FANCL depletion results in defective DNA replication restart following treatment with camptothecin, a drug that results in DSBs during DNA replication. This defect is more pronounced following treatment with mitomycin C, presumably because of an additional role of the FA pathway in DNA crosslink repair. Moreover, we show that chromatin binding of FA core complex proteins during DNA replication follows origin assembly and origin firing and is dependent on the binding of RPA to ssDNA while FANCD2 additionally requires ATR, consistent with FA proteins acting at replication forks. Together, our data suggest that FA proteins play a role in replication restart at collapsed replication forks.

| S-EPMC2596863 | biostudies-literature

Bioproduction of quercetin using recombinant thermostable glycosidases from Dictyoglomus thermophilum

Project description: Not available

| S-EPMC10991118 | biostudies-literature

Biochemical Characterization of Recombinant Thermostable Cohnella sp. A01 β-Glucanase

Project description:Background:Typically, non-cellulytic glucanase, including fungi and yeast cell wall hydrolyzing enzymes, are released by some symbiotic fungi and plants during the mycoparasitic fungi attack on plants. These enzymes are known as the defense mechanisms of plants. This study intends to investigate the biochemical properties of β-1,6-glucanase (bg16M) from native thermophilic bacteria, Cohnella A01. Methods:bg16M gene was cloned and expressed in E. coli BL21 (DE3). The enzyme was purified utilizing Ni-NTA nikcle sepharose column. Pustulan and laminarin were selected as substrates in enzyme assay. The purified bg16M enzyme was treated with different pH, temperature, metal ions, and detergents. Results:The expressed protein, including 639 amino acids, showed a high similarity with the hydrolytic glycosylated family 30. The molecular weight of enzyme was 64 kDa, and purification yield was 46%. The bg16M demonstrated activity as 4.83 U/ml on laminarin and 2.88 U/ml on pustulan. The optimum pH and temperature of the enzyme were 8 and 50 °C, respectively. The enzyme had an appropriate stability at high temperatures and in the pH range of 7 to 9, showing acceptable stability, while it did not lose enzymatic activity completely at acidic or basic pH. None of the studied metal ions and chemical compounds was the activator of bg16M, and urea, SDS, and copper acted as enzyme inhibitors. Conclusion:Biochemical characterization of this enzyme revealed that bg16M can be applied in beverage industries and medical sectors because of its high activity, as well as thermal and alkaline stability.

| S-EPMC6058188 | biostudies-literature

Membrane proteins enter the fold.

Project description:Membrane proteins have historically been recalcitrant to biophysical folding studies. However, recent adaptations of methods from the soluble protein folding field have found success in their applications to transmembrane proteins composed of both α-helical and β-barrel conformations. Avoiding aggregation is critical for the success of these experiments. Altogether these studies are leading to discoveries of folding trajectories, foundational stabilizing forces and better-defined endpoints that enable more accurate interpretation of thermodynamic data. Increased information on membrane protein folding in the cell shows that the emerging biophysical principles are largely recapitulated even in the complex biological environment.

| S-EPMC8405458 | biostudies-literature

Recombinant thermostable cycloinulo-oligosaccharide fructanotransferase produced by Saccharomyces cerevisiae.

Project description:A truncated fragment of the cycloinulo-oligosaccharide fructanotransferase (CFTase) gene of Bacillus circulans MCI-2554 was fused to the prepro secretion sequence of the alpha-factor and expressed in Saccharomyces cerevisiae under the control of the 5' upstream region of the isocitrate lyase gene of Candida tropicalis (UPR-ICL). Efficiently secreted recombinant CFTase protein (yeast CFTase) was purified. Yeast CFTase consisted of three protein molecules, each of which had CFTase activity (yeast CFTase 1 [116 kDa], yeast CFTase 2 [117 kDa], and yeast CFTase 3 [116 kDa]). Yeast CFTase 2 was the major product of the expression system employed and was shown to be N glycosylated by endoglycosidase H treatment. Yeast CFTase 1 was N glycosylated but had a short truncation at its N terminus, while yeast CFTase 3 did not contain an N-glycosylated carbohydrate chain(s). Yeast CFTase 2 showed an optimum pH, an optimum temperature, and a pH stability similar to those of CFTase purified from B. circulans but exhibited a significant increase in thermostability. Production of yeast CFTase by the strain which had two copies of the CFTase gene integrated into its chromosomes reached 391 U per liter of culture at 120 h, which corresponded to 8.40 mg of protein per liter, by shake-flask cultivation.

| S-EPMC168824 | biostudies-other

Nanostructured Mineral Coatings Stabilize Proteins for Therapeutic Delivery.

Project description:Proteins tend to lose their biological activity due to their fragile structural conformation during formulation, storage, and delivery. Thus, the inability to stabilize proteins in controlled-release systems represents a major obstacle in drug delivery. Here, a bone mineral inspired protein stabilization strategy is presented, which uses nanostructured mineral coatings on medical devices. Proteins bound within the nanostructured coatings demonstrate enhanced stability against extreme external stressors, including organic solvents, proteases, and ethylene oxide gas sterilization. The protein stabilization effect is attributed to the maintenance of protein conformational structure, which is closely related to the nanoscale feature sizes of the mineral coatings. Basic fibroblast growth factor (bFGF) released from a nanostructured mineral coating maintains its biological activity for weeks during release, while it maintains activity for less than 7 d during release from commonly used polymeric microspheres. Delivery of the growth factors bFGF and vascular endothelial growth factor using a mineral coated surgical suture significantly improves functional Achilles tendon healing in a rabbit model, resulting in increased vascularization, more mature collagen fiber organization, and a two fold improvement in mechanical properties. The findings of this study demonstrate that biomimetic interactions between proteins and nanostructured minerals provide a new, broadly applicable mechanism to stabilize proteins in the context of drug delivery and regenerative medicine.

| S-EPMC5628619 | biostudies-literature

Alveolar proteins stabilize cortical microtubules in Toxoplasma gondii.

Project description:Single-celled protists use elaborate cytoskeletal structures, including arrays of microtubules at the cell periphery, to maintain polarity and rigidity. The obligate intracellular parasite Toxoplasma gondii has unusually stable cortical microtubules beneath the alveoli, a network of flattened membrane vesicles that subtends the plasmalemma. However, anchoring of microtubules along alveolar membranes is not understood. Here, we show that GAPM1a, an integral membrane protein of the alveoli, plays a role in maintaining microtubule stability. Degradation of GAPM1a causes cortical microtubule disorganisation and subsequent depolymerisation. These changes in the cytoskeleton lead to parasites becoming shorter and rounder, which is accompanied by a decrease in cellular volume. Extended GAPM1a depletion leads to severe defects in division, reminiscent of the effect of disrupting other alveolar proteins. We suggest that GAPM proteins link the cortical microtubules to the alveoli and are required to maintain the shape and rigidity of apicomplexan zoites.

| S-EPMC6344517 | biostudies-literature

Extant fold-switching proteins are widespread.

Project description:A central tenet of biology is that globular proteins have a unique 3D structure under physiological conditions. Recent work has challenged this notion by demonstrating that some proteins switch folds, a process that involves remodeling of secondary structure in response to a few mutations (evolved fold switchers) or cellular stimuli (extant fold switchers). To date, extant fold switchers have been viewed as rare byproducts of evolution, but their frequency has been neither quantified nor estimated. By systematically and exhaustively searching the Protein Data Bank (PDB), we found ?100 extant fold-switching proteins. Furthermore, we gathered multiple lines of evidence suggesting that these proteins are widespread in nature. Based on these lines of evidence, we hypothesized that the frequency of extant fold-switching proteins may be underrepresented by the structures in the PDB. Thus, we sought to identify other putative extant fold switchers with only one solved conformation. To do this, we identified two characteristic features of our ?100 extant fold-switching proteins, incorrect secondary structure predictions and likely independent folding cooperativity, and searched the PDB for other proteins with similar features. Reassuringly, this method identified dozens of other proteins in the literature with indication of a structural change but only one solved conformation in the PDB. Thus, we used it to estimate that 0.5-4% of PDB proteins switch folds. These results demonstrate that extant fold-switching proteins are likely more common than the PDB reflects, which has implications for cell biology, genomics, and human health.

| S-EPMC6003340 | biostudies-literature

Fold combinations in multi-domain proteins.

Project description:Domain-domain interactions in multi-domain proteins play an important role in the combined function of individual domains for the overall biological activity of the protein. The functions of the tethered domains are often coupled and hence, limited numbers of domain architectures with defined folds are known in nature. Therefore, it is of interest to document the available fold-fold combinations and their preference in multi-domain proteins. Hence, we analyzed all multi-domain proteins with known structures in the protein databank and observed that only about 860 fold-fold combinations are present among them. Analyses of multi-domain proteins represented in sequence database result in recognition of 29,860 fold-fold combinations and it accounts for only 2.8% of the theoretically possible 1,036,080 (1439C2) fold-fold combinations. The observed preference for fold-fold combinations in multi-domain proteins is interesting in the context of multiple functions through structural adaptation by gene fusion.

| S-EPMC6589474 | biostudies-literature

How evolution makes proteins fold quickly.

Project description:Sequences of fast-folding model proteins (48 residues long on a cubic lattice) were generated by an evolution-like selection toward fast folding. We find that fast-folding proteins exhibit a specific folding mechanism in which all transition state conformations share a smaller subset of common contacts (folding nucleus). Acceleration of folding was accompanied by dramatic strengthening of interactions in the folding nucleus whereas average energy of nonnucleus interactions remained largely unchanged. Furthermore, the residues involved in the nucleus are the most conserved ones within families of evolved sequences. Our results imply that for each protein structure there is a small number of conserved positions that are key determinants of fast folding into that structure. This conjecture was tested on two protein superfamilies: the first having the classical monophosphate binding fold (CMBF; 98 families) and the second having type-III repeat fold (47 families). For each superfamily, we discovered a few positions that exhibit very strong and statistically significant "conservatism of conservatism"-amino acids in those positions are conserved within every family whereas the actual types of amino acids varied from family to family. Those amino acids are in spatial contact with each other. The experimental data of Serrano and coworkers [Lopez-Hernandez, E. & Serrano, L. (1996) Fold. Des. (London) 1, 43-55]. for one of the proteins of the CMBF superfamily (CheY) show that residues identified this way indeed belong to the folding nucleus. Further analysis revealed deep connections between nucleation in CMBF proteins and their function.

| S-EPMC20198 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data