Effect of protein fusion on the transition temperature of an environmentally responsive elastin-like polypeptide: a role for surface hydrophobicity?

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.     

Chemical Properties Determine Solubility and Stability in βγ-Crystallins of the Eye Lens

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.     

Intermediates in the protein folding process: a computational model

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.     

Hydrophobicity, solubility, and emulsifying properties of soy protein peptides prepared by papain modification and ultrafiltration

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 (S ₀), 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 S ₀, PS, EAI, and ESI were observed in the hydrolysates. Peptides in the permeates had higher PS and EAI but lower S ₀ 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.     

Effect of Alcalase™ on olive pomace protein extraction

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.     

融合标签在蛋白质可溶性表达中的应用进展

融合标签是大规模生产可溶性蛋白质的有效工具,因此融合标签在蛋白质可溶性表达中的应用已成为外源蛋白表达的研究热点。本文重点介绍了常用融合标签及其提高重组蛋白质可溶性表达的机理,主要用5种模型从不同的角度阐述融合标签在蛋白折叠中的作用,其中大多数功能较强的融合标签（如MBP和NusA）作为分子伴侣帮助重组蛋白正确折叠从而提高蛋白质可溶性。此外,阐述了实际应用中常用的组合标签和标签的去除。最后提出了融合标签在大规模生产可溶性蛋白质中的应用前景、面临的挑战,以及今后研究的方向。     

On the association between poly(vinyl alcohol) and sodium dodecyl sulfate and its effect on liquid–liquid interfacial tension: A mathematical model

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 (C_SDS), 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 C_SDS 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.     

What Macromolecular Crowding Can Do to a Protein

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.     

Nucleated polymerisation in the presence of pre-formed seed filaments

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.     

Extraction and properties of proteins in covercress™, new pennycress varieties developed as cover crop and alternative plant protein source

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.     

Fabrication and characterization of robust hydrophobic lotus leaf-like surface on Si3N4 porous membrane via polymer-derived SiNCO inorganic nanoparticle modification

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.     

Rapid Matrix‐Assisted Refolding of Histidine‐Tagged Proteins

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.