Protein-only solution

Having deposited a cake of yeast cells by filtering yeast only solutions (0.37 g L ) for 1 h, the feed was switched to protein-only solutions (50 mg L ). Maintaining a stable cake of yeast cells during the filtration of the protein-only solutions proved... 

The yield and the composition of the fractions from soy bean meal obtmned with isolating WUS is shown in Table 1. The removal of cold water solubles, proteins and starch from soy meal was successful. The larger part of the material appeared in CWS, 59.1%. UFF contained mainly oligosaccharides and some water soluble proteins and UFR contained mainly water soluble proteins. The solution of SDSS and DTT extracted the residual proteins from the soy meal and the extract consisted for over 80% of proteins. Since the yield of the HWS fraction is only 0.4%, the composition is not discussed here. The remaining WUS contained 90% of NSP and the yield was 15.7%, which indicates that from the polysaccharides present in soy meal 92% was recovered in the WUS. By isolating WUS a fraction is obtained in which almost all cell wall polysaccharides are recovered and which contained only little other components. 

The crystal structure of the OCP from Arthrospira maxima has been solved to 2.1 A resolution (Kerfeld et al. 2003). It is composed of two domains and the carotenoid, 3 -hydroxyechinenone, spans both. The carotenoid is almost completely buried within the protein only 3.4% of the pigment surface is accessible to solvent (see Figure 1.3a). The OCP is a dimer in solution the intermolecular interactions are largely mediated by hydrogen bonding among the N-terminal 30 amino acids, as shown in Figure 1.3b... 

In a fume hood, add 0.2 ml of 1 M sodium cyanoborohydride (Aldrich) to each ml of the protein/dextran solution. Mix well. Caution Cyanoborohydride is extremely toxic and should be handled only in well-ventilated fume hoods. Dispose of cyanide-containing solutions according to approved guidelines. 

Proteins have been studied for a long time. Beccari published an account of his experiments to isolate gluten in 1747 In 1805 Einhof discovered that a fraction of wheat gluten was soluble, while in 1858 Denis showed that many proteins of both plant and animal origin were soluble in saline solutions. In 1859 Ritthausen started to prepare highly purified proteins, only to be criticised by Weyl for using alkali to extract the proteins. Weyl in his work used the Denis method of extraction with neutral salts. 

These three examples emphasize the idea that a complete description of amyloid fibril polymorphisms will only be achieved when 3D structures at atomic detail become available (Luhrs et al., 2005). Last but not least, since the various morphologies observed for amyloid fibrils are defining the end point of the assembly process, an essential need is to properly define the early stages of fibril formation, namely the oligomeric states of the peptide or protein in solution prior to assembly into fibrils. 

Finally, the protein assay for the drug product will also be used for realtime and accelerated stability testing if it has been validated to be stability indicating. A stability-indicating protein concentration method usually translates to a method that can reveal how much protein can be recovered from the dosage form. Many protein instabilities result in precipitation of the protein and adsorption to the container. An instability that results in only a modification of the protein structure but not in loss of protein from solution will not be detected by a sequence-independent protein assay such as a colorimetric assay. 

The question can be raised as to whether the structure of the proteins within the ribosomal particle is the same as in the isolated state. The only direct evidence we have that the structures of proteins are not changed upon incorporation into the subunit is provided by the neutron-scattering studies of Nierhaus et al. (1983b). They showed that individual proteins in solution had radii of gyration indistinguishable from those obtained from their counterparts on the ribosomal subunits in the same buffers and under identical preparation conditions. 

Even a small protein consists of many hundreds of atoms. The primary aim of a structure determination is to place each of these atoms in space by determining the coordinates of each atom relative to a fixed coordinate system. The only techniques that can provide detailed information of this type are based on diffraction methods, and X-ray diffraction is the primary method. We must consider the meaning of the structures derived from these studies. Now X-ray diffraction can only be used to determine structures in crystals. One is, however, really concerned with the structure of proteins in solution, and it is therefore necessary to examine the difference between structure in the solid state and the solution state. We consider differences in general between these two states, and then differences in specific cases. To perform structural studies in solution, spectroscopic methods must be used. These methods are quite different from diffraction methods, being concerned with specific absorption or emission... 

The severity of agitation and separation determines whether the separated medium is considered to have proteins in solution (protein solubility values) or in dispersion (protein dispersion values). The terms Nitrogen Solubility Index (NSI) and Protein Dispersibility Index (PDI) are to be used only when the official... 

But where there is an equilibrium among two or more conformations of the enzyme in solution, crystallization may select out only one of the conformations. a-Chymotrypsin has a substantial fraction of an inactive conformation present under the conditions of crystallization, but only the active form of the enzyme crystallizes. An allosteric effector molecule that changes the conformation of the protein in solution may have no effect on the crystalline protein, as, for example, with phosphorylase b.5A The enzyme is frozen in one conformation, with the crystal lattice forces preventing any conformational change. On the other hand, the addition of an effector to phosphorylase a causes the crystals first to crack and then to anneal, giving crystals of the enzyme in a second conformation. 

Depending on the fit, the number and strength of contacts, and other factors, a monomer-oligomer equilibrium will exist. For example, in the case of Concanavalin A the tetramer is in equilibrium with dimer and monomer under normal conditions. In the case of insulin the monomer, dimer, and hexamer are all in equilibrium. In hemoglobin the dimer and tetramer are in equilibrium. In the case of the adsorption of such proteins, one must not only know what is the aggregation state of the protein in solution, but must be able to deduce the adsorbed state. Do the molecules adsorb as dimers 21) Do they adsorb as dimers and then associate as tetramers or hexamers on the surface, etc. ... 

Because high levels of display can impair phage infection, we recommend treating the phages with 10-7 M trypsin for 30 min before measuring the titer. Note that trypsin removes the displayed protein only if a cleavable linker is used or if the protein itself is degraded by trypsin. The stock solution of trypsin (10-5 M) should be freshly prepared in 20 mM acetate buffer, pH 3.0. 

The reaction is carried out in 0.2 M triethanolamine, pH 8.2. DMS should be the limiting reagent in the reaction to avoid blocking all amines on both molecules with only one end of the cross-linker, thus eliminating any conjugation. The amounts of total lipid and protein in solution may have to be adjusted to optimize each conjugation reaction and avoid precipitation of protein or aggregation of liposomes. 

In order to test whether our CIRcle cell spectra were dominated by adsorbed protein or protein in solution, we ran spectra of a series of lysozyme solutions ranging in concentration from 0.12 to 102. The IR response of the amide I and II bands at 1653 and 1543 cm-1 is nearly linear with concentration between 5 and 102 lysozyme. However, the IR intensities change very little between 0.1 and 12, strongly suggesting that most of the signal we observe at 0.12 concentration is due to adsorbed lysozyme. Since our study of subtilisin BPN was done at 0.012, we are almost certainly observing only adsorbed species in our ATR spectra. 

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