Membrane protein extract materials

One area of rapidly expanding interest is the use of reverse micellar systems of sugar-based surfactants in the extraction of proteins and other sensitive materials. The use of hydrophilic, nonionic, sugar-based surfactants for membrane protein extraction is well known to be effective due to the mild, nondenaturing properties of these surfactants when compared with ionic surfactants or polyoxyethylene derivatives. For the same reasons, protein extraction into reverse micellar systems is now becoming a popular medium for such applications. Alkyl sorbitan esters and ethoxylated sorbitan esters, such as Tween 85 [107] and Span 60 [108], have been used successfully to form reverse micellar systems for protein extraction. Blends of Tween and Span have also been found to be effective for this purpose [109]. More recently, commercially available sucrose fatty acid esters have been shown to form biocompatible reverse micellar systems into which cytochrome c is effectively extracted [110]. 

Protegrin derivatives, 18 260 Protegrins, 18 260-261 properties of, 18 261 Protein. See also Proteins extraction of, 26 474 in cereal grains, 26 275-276 Proteinaceous materials, as membrane foulants, 21 664 Protein adsorption, 12 136-137 Protein affinity libraries, 12 516-517 Protein-based chiral phases, 6 89-90 Protein-based microarrays, 16 382 Protein biosynthesis, 20 450... 

Direct protein sequencing of the large proteins which constituted the channel was not possible both because of the very limited amount of material available and because of the not unexpected very hydrophobic nature of these membrane proteins. However short sequences from the amino terminal were obtained and these were sufficient to allow synthesis of degenerate oligonucleotide probes which were then used to probe a library of cDNA synthesized from a small quantity of mRNA extracted from the electric organ of the eel. The cDNA from the positive plasmids identified in this way was then sequenced and the primary amino acid sequence of all the subunits so obtained. 

Because of its occurence in diseased tissue, the mode of association of cholesterol esters with biomembranes is of interest. Possible modes of association could be droplets within the hydrophobic core of the membrane bilayer, binding to membrane protein or as part of membrane attached serum lipoproteins. A potentially useful model system for investigating this association is the membrane of the microorganism Mycoplasma capricolum. The Mycoplasma due to their simplicity have served as model membrane systems in many studies. As mentioned previously, cholesterol esters show complex behavior that is a function of thermal history, impurities and physical packing constraints. Using DSC on native membranes and extracted membrane material, it was possible to demonstrate that the majority of cholesterol esters associated with the membranes of M. capricolum exist as relatively large and pure liquid droplets (17). 

Glycosylurea and thiourea derivatives, e.g. (19), were synthesized by condensation of alkyl isocyanates and isothiocyanates respectively, with jS-D-glucopyranosylamine and fi-lactosylamine, as non-ionic detergents of particular interest for extraction of membrane proteins. Various l-( -D-glycosyl)-derivatives of diphenylhydantoin and hydrochlorothiazide were synthesized in an attempt to enhance the hydrophilicity and biological activity of these materials. Conformation analysis and X-ray structures of some 1,2- and 1,3-fused glycosylamine derivatives are included in Chapters 21 and 22. 

Membrane-retained components are collectively called concentrate or retentate. Materials permeating the membrane are called filtrate, ultrafiltrate, or permeate. It is the objective of ultrafiltration to recover or concentrate particular species in the retentate (eg, latex concentration, pigment recovery, protein recovery from cheese and casein wheys, and concentration of proteins for biopharmaceuticals) or to produce a purified permeate (eg, sewage treatment, production of sterile water or antibiotics, etc). Diafiltration is a specific ultrafiltration process in which the retentate is further purified or the permeable sohds are extracted further by the addition of water or, in the case of proteins, buffer to the retentate. 

The important bacterial storage material poly-hydroxybutyric acid is related metabolically and structurally to the lipids. This highly reduced polymer is made up of D-(3-hydroxybutyric acid units in ester linkage, about 1500 residues being present per chain. The structure is that of a compact right-handed coil with a twofold screw axis and a pitch of 0.60 nm.a Within bacteria it often occurs in thin lamellae 5.0 nm thick. Since a chain of 1500 residues stretches to 440 nm, there must be 88 folds in a single chain. Present in both cytoplasmic granules and in membranes,b polyhydroxybutyrate can account for as much as 50% of the total carbon of some bacterial In E. coli and many other bacteria polyhydroxybutyrate is present in a lower molecular mass form bound to calcium polyphosphates, proteins, or other macromolecules.d e It has also been extracted from bovine serum albumin and may be ubiquitous in both eukaryotes and prokaryotes.d/e The polymer may function in formation of Ca2+ channels in membranes.b/d... 

---