The limited throughput, scalability and high cost of protein purification by chromatography provide motivation for the development of non-chromatographic protein purification technologies that are cheaper and easier to implement in a high-throughput format for proteomics applications and to scale up for industrial bioprocessing. We have shown that genetic fusion of a recombinant protein to an elastin-like polypeptide (ELP) imparts the environmentally sensitive solubility property of the ELP to the fusion protein, and thereby allows selective separation of the fusion protein from Escherichia coli lysate by aggregation above a critical temperature (T(t)). Further development of ELP fusion proteins as widely applicable purification tools necessitates a quantitative understanding of how fused proteins perturb the ELP T(t) such that purification conditions (T(t)) may be predicted a priori for new recombinant proteins. We report here the effect that fusing six different proteins has on the T(t) of an ELP. A negative correlation between T(t) and the fraction hydrophobic surface area on the fused proteins was observed, which was determined from computer modeling of the available three-dimensional structure. The thermally triggered aggregation behavior of ELP-coated, functionalized gold colloids as well as ligand binding to the tendamistat-ELP fusion protein support the hypothesis that hydrophobic surfaces in molecular proximity to ELPs depress the ELP T(t) by a mechanism analogous to hydrophobic residue substitution in the ELP repeat, Val-Pro-Gly-Xaa-Gly. 相似文献
βγ-Crystallins are the primary structural and refractive proteins found in the vertebrate eye lens. Because crystallins are not replaced after early eye development, their solubility and stability must be maintained for a lifetime, which is even more remarkable given the high protein concentration in the lens. Aggregation of crystallins caused by mutations or post-translational modifications can reduce crystallin protein stability and alter intermolecular interactions. Common post-translational modifications that can cause age-related cataracts include deamidation, oxidation, and tryptophan derivatization. Metal ion binding can also trigger reduced crystallin solubility through a variety of mechanisms. Interprotein interactions are critical to maintaining lens transparency: crystallins can undergo domain swapping, disulfide bonding, and liquid-liquid phase separation, all of which can cause opacity depending on the context. Important experimental techniques for assessing crystallin conformation in the absence of a high-resolution structure include dye-binding assays, circular dichroism, fluorescence, light scattering, and transition metal FRET. 相似文献
The paper presents a model for simulating the protein folding process in silico. The two-step model (which consists of the early stage-ES and the late stage-LS) is verified using two proteins, one of which is treated (according to experimental observations) as the early stage and the second as an example of the LS step. The early stage is based solely on backbone structural preferences, while the LS model takes into account the water environment, treated as an external hydrophobic force field and represented by a 3D Gauss function. The characteristics of 1ZTR (the ES intermediate, as compared with 1ENH, which is the LS intermediate) confirm the link between the gradual disappearance of ES characteristics in LS structural forms and the simultaneous emergence of LS properties in the 1ENH protein. Positive verification of ES and LS characteristics in these two proteins (1ZTR and 1ENH respectively) suggest potential applicability of the presented model to in silico protein folding simulations. 相似文献
Peptide size control is important for obtaining desirable functional properties so that these peptides can be better utilized.
Proteolytic enzymatic modification of soy protein isolates (SPI), followed by ultrafiltration, is an effective way to fractionate
these proteins into peptides with controlled molecular size. SPI was predenatured by mild alkali at pH 10 and heated at 50°C
for 1 h prior to partial hydrolysis by papain at pH 7.0 and 38°C for 10, 30, and 60 min (PMSPI10, PMSPI30, and PMSPI60). The
hydrolysate PMSPI60 was further fractionated by ultrafiltration with a stirred cell and disc membranes (100-, 50-, and 20-kDa
molecular weight cut-off) into one retentate (R100) and three permeates (P100, P50, and P20). Molecular weight distribution,
surface hydrophobicity (S0), protein solubility (PS), emulsifying activity index (EAI), and emulsion stability index (ESI) of the control SPI (without
added papain), hydrolysates, and ultrafiltrates were investigated. Significant increases (P<0.001) in S0, PS, EAI, and ESI were observed in the hydrolysates. Peptides in the permeates had higher PS and EAI but lower S0 than the peptides in the retentate and hydrolysate. Soy protein peptides that were prepared from SPI by papain modification
and ultrafiltration had lower molecular weight, higher solubility, and higher emulsifying properties. They could find use
in products that require these properties, especially in the cosmetic and health food industries. 相似文献
Prion protein aggregation into amyloid fibrils is associated with the onset and progression of prion diseases—a group of neurodegenerative amyloidoses. The process of such aggregate formation is still not fully understood, especially regarding their polymorphism, an event where the same type of protein forms multiple, conformationally and morphologically distinct structures. Considering that such structural variations can greatly complicate the search for potential antiamyloid compounds, either by having specific propagation properties or stability, it is important to better understand this aggregation event. We have recently reported the ability of prion protein fibrils to obtain at least two distinct conformations under identical conditions, which raised the question if this occurrence is tied to only certain environmental conditions. In this work, we examined a large sample size of prion protein aggregation reactions under a range of temperatures and analyzed the resulting fibril dye-binding, secondary structure and morphological properties. We show that all temperature conditions lead to the formation of more than one fibril type and that this variability may depend on the state of the initial prion protein molecules. 相似文献
Prion diseases are associated with conformational conversion of cellular prion protein into a misfolded pathogenic form, which resembles many properties of amyloid fibrils. The same prion protein sequence can misfold into different conformations, which are responsible for variations in prion disease phenotypes (prion strains). In this work, we use atomic force microscopy, FTIR spectroscopy and magic-angle spinning NMR to devise structural models of mouse prion protein fibrils prepared in three different denaturing conditions. We find that the fibril core region as well as the structure of its N- and C-terminal parts is almost identical between the three fibrils. In contrast, the central part differs in length of β-strands and the arrangement of charged residues. We propose that the denaturant ionic strength plays a major role in determining the structure of fibrils obtained in a particular condition by stabilizing fibril core interior-facing glutamic acid residues. 相似文献
The effect of Alcalase™ treatment on olive pomace protein extraction has been studied. Alcalase improves protein extraction from 5 to 30% of total protein. This improvement is not accompained by an increase in degree of protein hydrolysis, probably because protease activity is inhibited by secondary metabolites and the substrate is highly denatured and resistant to hydrolysis. The increase in protein extraction is attributed to fiber solubilization as a result of secondary activities of Alcalase. Protein extracts with a high content of soluble fiber have improved functional properties, with respect to olive pomace, such as water and oil absorption. Emulsifying and foaming activities were inappreciable. The products obtained represent a suitable source of soluble fiber and protein and may contribute to the improvement of the economic status of olive pomace by-product. 相似文献
p62/Sequestosome-1 (p62) is a multifunctional adaptor protein and is also a constant component of disease-associated protein aggregates, including Mallory–Denk bodies (MDBs), in steatohepatitis and hepatocellular carcinoma. We investigated the interaction of the two human p62 isoforms, p62-H1 (full-length isoform) and p62-H2 (partly devoid of PB1 domain), with keratins 8 and 18, the major components of MDBs. In human liver, p62-H2 is expressed two-fold higher compared to p62-H1 at the mRNA level and is present in slightly but not significantly higher concentrations at the protein level. Co-transfection studies in CHO-K1 cells, PLC/PRF/5 cells as well as p62− total-knockout and wild-type mouse fibroblasts revealed marked differences in the cytoplasmic distribution and aggregation behavior of the two p62 isoforms. Transfection-induced overexpression of p62-H2 generated large cytoplasmic aggregates in PLC/PRF/5 and CHO-K1 cells that mostly co-localized with transfected keratins resembling MDBs or (transfection without keratins) intracytoplasmic hyaline bodies. In fibroblasts, however, transfected p62-H2 was predominantly diffusely distributed in the cytoplasm. Aggregation of p62-H2 and p62ΔSH2 as well as the interaction with K8 (but not with K18) involves acquisition of cross-β-sheet conformation as revealed by staining with luminescent conjugated oligothiophenes. These results indicate the importance of considering p62 isoforms in protein aggregation disease. 相似文献
Association between poly(vinyl alcohol-co-vinyl acetate) copolymer (PVA) and sodium dodecyl sulfate (SDS) was studied experimentally and theoretically. It was found that, for the ethyl acetate-aqueous phase interface in which PVA was previously adsorbed, the interfacial tension (γ) increases abruptly to a maximum and then exhibits a relatively mild decay with the addition of SDS to the aqueous phase. The theoretical results indicate that vinyl acetate (VAc) segments determine γ. However, for relatively low concentrations of SDS (CSDS), this latter plays a major role because through its association with the VAc segments it modulates the extent to which PVA is adsorbed at the interface, indirectly determining the value of γ. As CSDS approaches to the CMC value for SDS, its influence on γ decreases because SDS tends to self-assembly rather than associates with VAc. These model predictions are consistent with experimental findings reported in the literature. 相似文献
The intracellular environment represents an extremely crowded milieu, with a limited amount of free water and an almost complete lack of unoccupied space. Obviously, slightly salted aqueous solutions containing low concentrations of a biomolecule of interest are too simplistic to mimic the “real life” situation, where the biomolecule of interest scrambles and wades through the tightly packed crowd. In laboratory practice, such macromolecular crowding is typically mimicked by concentrated solutions of various polymers that serve as model “crowding agents”. Studies under these conditions revealed that macromolecular crowding might affect protein structure, folding, shape, conformational stability, binding of small molecules, enzymatic activity, protein-protein interactions, protein-nucleic acid interactions, and pathological aggregation. The goal of this review is to systematically analyze currently available experimental data on the variety of effects of macromolecular crowding on a protein molecule. The review covers more than 320 papers and therefore represents one of the most comprehensive compendia of the current knowledge in this exciting area. 相似文献
We revisit the classical problem of nucleated polymerisation and derive a range of exact results describing polymerisation in systems intermediate between the well-known limiting cases of a reaction starting from purely soluble material and for a reaction where no new growth nuclei are formed. 相似文献
Parkinson’s disease (PD) is the second most common neurodegenerative disorder. An important hallmark of PD involves the pathological aggregation of proteins in structures known as Lewy bodies. The major component of these proteinaceous inclusions is alpha (α)-synuclein. In different conditions, α-synuclein can assume conformations rich in either α-helix or β-sheets. The mechanisms of α-synuclein misfolding, aggregation, and fibrillation remain unknown, but it is thought that β-sheet conformation of α-synuclein is responsible for its associated toxic mechanisms. To gain fundamental insights into the process of α-synuclein misfolding and aggregation, the secondary structure of this protein in the presence of charged and non-charged surfactant solutions was characterized. The selected surfactants were (anionic) sodium dodecyl sulphate (SDS), (cationic) cetyltrimethylammonium chloride (CTAC), and (uncharged) octyl β-D-glucopyranoside (OG). The effect of surfactants in α-synuclein misfolding was assessed by ultra-structural analyses, in vitro aggregation assays, and secondary structure analyses. The α-synuclein aggregation in the presence of negatively charged SDS suggests that SDS-monomer complexes stimulate the aggregation process. A reduction in the electrostatic repulsion between N- and C-terminal and in the hydrophobic interactions between the NAC (non-amyloid beta component) region and the C-terminal seems to be important to undergo aggregation. Fourier transform infrared spectroscopy (FTIR) measurements show that β-sheet structures comprise the assembly of the fibrils. 相似文献
Pennycress (Thlaspi arvense L.) is a winter oilseed crop that also functions as cover crop, oil feedstock for biofuel, and novel plant-based protein. New pennycress specialty varieties were developed by conventional breeding techniques, but protein chemical and functional traits are still unknown. This research evaluated three new lines (black-seeded B3 and yellow-seeded TTG1/Y1126 and TT8) for protein extractability and functionality. Seeds were ground cryogenically and then hexane-defatted until residual oil content was 0.5%–0.7% dry basis, db. Protein in defatted seed meals was extracted using saline method (1: 10 wt/vol, 0.1 M NaCl, 2 h, 50°C). TT8 and Y1126 had greater protein recoveries (ca. 50%) than those of B3 (36%) and wild-type pennycress (WTP, 40%). TT8 produced higher purity protein isolate (94.3%) than did Y1126 (75.0%) and B3 (71.3%). TT8 and Y1126 protein band patterns were different from WTP and B3, reflecting varietal changes. The new varieties' protein extracts were more soluble than WTP protein in acidic pH, with TT8 showing the highest solubility (81%–93%). TT8, Y1126 and B3 foaming capacities (113–135 ml) were superior to those of WTP (101–107 ml). Emulsification properties (EAI 95–230 m2/g protein, ESI 13–24 min) were similar and increased with pH. B3, Y1126, and TT8 formed heat-induced gels at much lower concentration than did WTP (3% vs. 6%) and their water-holding capacities (pH 7) were two to fivefold greater. This work demonstrated that mutations in the TTG1 or TT8 gene in yellow-seeded lines resulted in improved protein recoveries and functionalities that are desirable in plant-based proteins. 相似文献
Robust hydrophobic surface was produced by modifying the surface of porous Si3N4 membrane, via aminolysis and pyrolysis process, with organosilane-derived inorganic SiNCO nanoparticles, which are tightly adhered to the Si3N4 grains. The resultant material had a high water contact angle of 142°, attributed to -Si-CH3 surface terminal groups and a lotus leaf-like hierarchical structure of the nanoparticles, which had a frame structure with Si-N and Si-O covalent bonds in their bulk. The hydrophobic behavior remained practically unchanged after exposure of the produced membranes to aqueous solutions of humic acid, HCl and NaOH, to benzene, as well as to stirring abrasive slurry with SiC particles, and after exposure at high temperatures, up to 500?°C, to air. The inorganic membrane can be considered for use in a broad range of applications which require robust hydrophobic surfaces. 相似文献
Nothofagin is a prominent bioactive ingredient of rooibos tea. We recently reported its synthesis through a glucosyltransferase cascade reaction involving 3′‐C‐β‐D ‐glucosylation of the dihydrochalcone phloretin from uridine 5′‐diphosphate (UDP)‐glucose and in situ formation of UDP‐glucose from sucrose and catalytic amounts of UDP. Here we show that the limitation in process efficiency caused by the vanishingly low water solubility of phloretin – a major problem for biocatalytic modifications of hydrophobic natural products in general – was overcome effectively using phloretin inclusion complexation with β‐cyclodextrin. Unlike operating in a two‐phase system containing uncomplexed insoluble phloretin or using organic cosolvents, the addition of β‐cyclodextrin inclusion complexes was well tolerated regarding enzyme activity and stability. Besides enhancing the effective phloretin concentration in water (∼0.2 mM) to about 50 mM , inclusion complexation offered the additional advantage of overcoming the complex inhibition/inactivation effect of the free/microaggregated dihydrochalcone acceptor. Thus oversaturated phloretin solution was transformed in a single batch reaction in excellent conversion (99% in solution; 88% overall) and isolated yield (78%; 17.0 g L −1). The UDP‐glucose was regenerated up to ∼90 times and the nothofagin space‐time yield of 2.4 mM h−1 presented an eight‐fold improvement compared to a reference reaction using 20% DMSO (dimethyl sulfoxide) and requiring controlled phloretin feed. We thus demonstrate the high potential of inclusion complexation by cyclodextrins for boosting the glycosylation of hydrophobic flavonoid‐like natural products.
Matrix refolded : The formation of inclusion bodies, which are amorphous aggregates of misfolded insoluble protein, during recombinant protein expression, is one of the biggest bottlenecks in protein science. We report a stepwise, rational optimization procedure for refolding of insoluble proteins (see scheme). In comparison to refolding in‐solution, this parallelized, matrix‐assisted approach allows the refolding of various proteins in a fast and efficient manner.
