Pure Protein (pure + protein)

Distribution by Scientific Domains

Selected Abstracts

Characterization of amorphous solids with weak glass transitions using high ramp rate differential scanning calorimetry

Derrick S. Katayama
Abstract Measurement of the glass transition temperature (Tg) of proteins and other high molecular weight polymers in the amorphous state is often difficult, since the transition is extremely weak, that is, the ,Cp at the glass transition temperature is small. For example, little is known about the solid-state properties of hydroxyethyl starch (HES), which is beginning to become more commonly evaluated as a bulking agent in pharmaceutical products. For weak thermal events, such as the change in heat capacity at the Tg of a pure protein or large synthetic polymer, increased heating rate should produce greater sensitivity in terms of heat flow. Recent innovations in rapid scanning technology for differential scanning calorimetry (DSC) allow measurements on materials where the thermal events are difficult to detect by conventional DSC. In the current study, measurements of the Tg of proteins in the solid state, amorphous pharmaceutical excipients which have small ,Cp at the glass transition temperature, and bacterial spores, have all been made using high ramp rate DSC, providing information on materials that was inaccessible using conventional DSC methods. 2007 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 97:1013,1024, 2008 [source]

Purification and expression of a protein elicitor from Alternaria tenuissima and elicitor-mediated defence responses in tobacco

J. Mao
A new protein elicitor, PeaT1, was purified from the mycelium of Alternaria tenuissima by column chromatography. PeaT1 was identified as a heat-stable and acidic protein. It induced systemic acquired resistance to tobacco mosaic virus (TMV) in tobacco plants but did not cause hypersensitive response. The elicitor-encoding gene was cloned by rapid amplification of cDNA ends method. Sequence analysis revealed that the cDNA is 624 bp in length and the open reading frame encodes for a polypeptide of 207 amino acids with a nascent polypeptide-associated complex domain. The peaT1 gene was cloned into the expression vector pET-28a and transformed into Escherichia coli BL21 (DE3). The recombinant elicitor also triggered defence responses in intact tobacco plants. The availability of the pure protein offers the possibility to isolate the corresponding receptor and links it to the downstream signalling pathway. [source]

Interaction between diet and genetic aptitude for weight and growth in juvenile rainbow trout, Oncorhynchus mykiss (Walbaum)

J M Blanc
Abstract Samples of rainbow trout, Oncorhynchus mykiss (Walbaum) alevins from 17 independent families (full-sib-groups) were raised from the start of feeding until the age of 18 weeks post-hatching with three diets (C, G and S) differing in protein content (fish soluble protein concentrate: 84% in C, 54% in G and 44% in S) and carbohydrates (none in C, 30% glucose in G and 40% crude corn starch in S). Fish were fed to near satiation, and their body weight and growth were measured. Diet effect was highly significant (G<,C < S), as well as the familial effect. The major part of the familial variance (80,90%) was common to the three diets. However, a minor part of the familial variance was observed to be diet dependent (family diet interaction), and was found to result mainly from relative performances with carbohydrates (G and S diets) vs. pure protein (C diet). These results indicate that genetic improvement of growth should suffer little impairment from possible changes in future feed formulations. [source]

When less is more: a more efficient vapour-diffusion protocol

Kirsty V. Dunlop
Reducing protein consumption during crystallization screening is of utmost importance to crystallographers because of the time, effort and money that goes into producing pure protein. One approach is to reduce sample volumes with robotics, but a patent and the high cost of equipment limits access. Here, it is shown that the same result can be obtained by reducing the sample concentration in a modified vapour-diffusion protocol, the dilution method. In this protocol, the protein and mother liquor in the crystallization drop are both diluted, while the mother liquor in the well remains undiluted. Vapour diffusion will shrink the initial volume of the crystallization drop, e.g. 1,l or more, to a drop size equivalent to one dispensed by a robot. This new crystallization method circumvents some of the current problems associated with robotic crystallization screening trials. Because of the large initial volume of the crystallization drop, the evaporation problem is eliminated and dispensing accuracy is improved. In addition, the likelihood that the crystallization experiment starts in the undersaturated region is increased. [source]

Preliminary structural investigations of the Eut-L shell protein of the ethanolamine ammonia-lyase metabolosome of Escherichia coli

Kiel Nikolakakis
The ethanolamine ammonia-lyase microcompartment is composed of five different shell proteins that have been proposed to assemble into symmetrically shaped polyhedral particles of varying sizes. Here, preliminary X-ray analysis of crystals of the bacterial microcompartment shell protein Eut-L from Escherichia coli is reported. Cloning, overexpression and purification resulted in highly pure protein that crystallized readily under many different conditions. In all cases the protein forms thin hexagonal plate-shaped crystals belonging to space group P3 that are of unusually high stability against different solvent conditions. The crystals diffracted to a resolution of 2.0, using synchrotron radiation but proved to be radiation-sensitive. Preparations of heavy-atom-derivatized crystals for use in determining the three-dimensional structure are under way. [source]

Disulfiram is an Inhibitor of Human Purified Monoacylglycerol Lipase, the Enzyme Regulating 2-Arachidonoylglycerol Signaling

CHEMBIOCHEM, Issue 11 2007
Geoffray Labar
Abstract Monoacylglycerol lipase (MAGL) is a key enzyme responsible for the termination of endocannabinoid signaling. Its crucial role in 2-arachidonoylglycerol (2-AG) metabolism, together with the numerous pharmacological properties mediated by this endocannabinoid, emphasize the interest in MAGL as therapeutic target, along with the need to design potent and selective inhibitors. Meanwhile, the complexity of 2-AG degradation pathways underscores the need to use a purified source of enzyme in evaluation studies of new inhibitors. We report here the first heterologous expression and purification of human MAGL. A highly pure protein was obtained and allowed us to measure the affinity of several MAGL inhibitors for the human enzyme. Importantly, disulfiram (tetraethylthiuram disulfide), a compound used to treat alcoholism, and other disulfide-containing compounds were shown to inhibit MAGL with good potency, likely through an interaction with cysteine residues. [source]

Tailoring orthogonal proteomic routines to understand protein separation during ion exchange chromatography

Rosa Cabrera
Abstract Surface charge, molecular weight, and folding state are known to influence protein chromatographic behaviour onto ion exchangers. Experimentally, information related to such factors can be gathered via 2-DE methods. The application of 2-D PAGE under denaturing/reducing conditions was already shown to reveal separation trends within a large protein population from cell extracts. However, ion-exchange chromatography normally runs under native conditions. A tailored protocol consisting in a first separation based on IEF on ImmobilineTM strips under native conditions followed by a second dimension SDS-PAGE run was adopted. The chromatographic versus electrophoretic separation behaviours of two model proteins, thaumatin (TAU) and BSA, were compared to better understand which proteomic routine would be better suited to anticipate IEX chromatographic separations. It was observed that the information contained in the pI value obtained with the adapted 2-DE protocol showed better correlation with the IEX chromatographic behaviour. On the other hand, chromatographic separations performed in the presence of urea as a denaturant have demonstrated the potential influence of hydrodynamic radius/conformation on protein separation. Moreover, the information provided by such 2-D system correlated well with the chromatographic behaviour of an additional set of pure proteins. An initial prediction of protein ion-exchange chromatographic behaviour could be possible utilizing an experimental approach based on 2-DE running under milder chemical conditions. This technique provides information that more closely resembles the separation behaviour observed with a complex biotechnological feedstock. [source]