Protein permeation

K Burczak, T Fujisato, M Hatada, Y Ikada. Protein permeation through polymer membranes for hybrid-type artificial pancreas. Proc Jpn Acad B 67 83-88, 1991. 

The large (SOS) ribosomal subunit is where protein synthesis, catalyzed formation of peptide bonds, takes place. In light of the discoveries by Thomas Cech and Sidney Altman of ribozymes, RNA molecules that behave as enzymes, the question of catalysis of protein synthesis by RNA or proteins in the ribosome was a major one. The 3 OS ribosomal subunit is where transfer RNAs bind with ribosomal RNA codons. The 50S ribosomal subunit can be further separated into a 23S secondary subunit and a smaller 5S secondary subunit that are normally held together by protein molecules. The 23S subunit includes 3045 nucleotides and 31 proteins. There are six discrete RNA domains in the S23 unit particle. The 5S unit effectively adds a seventh domain. The proteins permeate the exterior of the RNA of S23, but are located over 18 A distant from the active site of catalytic protein bond formation. The structures of complexes of the S23 subunit with two inactive substrate molecules suggest mechanistic similarities to the enzyme chymotrypsin. The S23 structure indicates that an adenine base at the active site plays a role analogous to that of histidine-... 

A 1% protein solution = 1 ro ) is concentrated in a batch process to 1/15 of the original volume. Calculate the protein permeate concentration in a perfect mixed system and a plug flow system at the feed side. The protein retention is 99%. 

The fluorescence approach discussed provides information that may contribute to the development of new membrane materials more suitable for protein permeation, in terms of their surface chemistry and structural characteristics with the purpose of minimizing the negative impact of processing on protein function. 

Target proteins permeating through the membrane are captured by the beads... 

Aqueous Interfaces Biomembranes Modeling CHAR-MM The Energy Function and Its Parameterization Hydrophobic Effect Molecular Dynamics Studies of Lipid Bilayers Molecular Dynamics Techniques and Applications to Proteins Permeation of Lipid Membranes Molecular Dynamics Simulations Protein Force Fields. 

Gel-permeation media are extremely versatile and may be used for separation of particles such as vimses (Fig. 11) as well as proteins (34). Separations of proteins and other particles having sizes equivalent to a molecular weight of 40 x 10 are possible using the agar-based Sepharose-type gel. This particular gel has a limited temperature range for operation, however. It melts upon heating to 40°C (34). 

Films or membranes of silkworm silk have been produced by air-drying aqueous solutions prepared from the concentrated salts, followed by dialysis (11,28). The films, which are water soluble, generally contain silk in the silk I conformation with a significant content of random coil. Many different treatments have been used to modify these films to decrease their water solubiUty by converting silk I to silk II in a process found usehil for enzyme entrapment (28). Silk membranes have also been cast from fibroin solutions and characterized for permeation properties. Oxygen and water vapor transmission rates were dependent on the exposure conditions to methanol to faciUtate the conversion to silk II (29). Thin monolayer films have been formed from solubilized silkworm silk using Langmuir techniques to faciUtate stmctural characterization of the protein (30). ResolubiLized silkworm cocoon silk has been spun into fibers (31), as have recombinant silkworm silks (32). 

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. 

Electroultrafiltration has been demonstrated on clay suspensions, electrophoretic paints, protein solutions, oil—water emulsions, and a variety of other materials. Flux improvement is proportional to the appHed electric field E up to some field strength E where particle movement away from the membrane is equal to the Hquid flow toward the membrane. There is no gel-polarization layer and (in theory) flux equals the theoretical permeate flux. It... 

Second, most membrane materials adsorb proteins. Worse, the adsorption is membrane-material specific and is dependent on concentration, pH, ionic strength, temperature, and so on. Adsorption has two consequences it changes the membrane pore size because solutes are adsorbed near and in membrane pores and it removes protein from the permeate by adsorption in addition to that removed by sieving. Porter (op. cit., p. 160) gives an illustrative table for adsorption of Cytochrome C on materials used for UF membranes, with values ranging from 1 to 25 percent. Because of the adsorption effects, membranes are characterized only when clean. Fouling has a dramatic effect on membrane retention, as is explained in its own section below. 

Diafiltration If a batch process is run so that the permeate is replaced by an equal volume of fresh solvent, unretained solutes are flushed through the system more efficiently. A major use of UF is fractionation, where a solvent, a retained solute and an unretained solute are present. An example is whey, containing water, protein, and lactose. If the retention of protein is I and the retention of lactose is 0, the concentration of protein in the retentate rises during UF. The ratio of protein to lac tose rises, but the feed concentration of lactose is unchanged in retentate and permeate. Diafiltration dilutes the feed, and permits the concentration of lactose to be reduced. Diafiltration is used to produce high-purity products, and is used to fractionate high-value products. R is always 0 for eveiy component. 

Size exclusion was first noted in the late fifties when separations of proteins on columns packed with swollen maize starch were observed (Lindqvist and Storgards, 1955 Lathe and Ruthven, 1956). The run time was typically 48 hr. With the advent of a commercial material for size separation of molecules, a gel of cross-linked dextran, researchers were given a purposely made material for size exclusion, or gel filtration, of solutes as described in the classical work by Porath and Flodin (1959). The material, named Sephadex, was made available commercially by Pharmacia in 1959. This promoted a rapid development of the technique and it was soon applied to the separation of proteins and aqueous polymers. The work by Porath and Flodin promoted Moore (1964) to apply the technique to size separation, gel permeation chromatography of organic molecules on gels of lightly cross-linked polystyrene (i.e., Styragel). 

H. Yamamoto, T. Manabe and T. Okuyama, Gel permeation chromatography combined with capillary electrophoresis for microanalysis of proteins , 7. Chromatogr. 480 277-283 (1989). 

Ultrafiltration of micellar solutions combines the high permeate flows commonly found in ultrafiltration systems with the possibility of removing molecules independent of their size, since micelles can specifically solubilize or bind low molecular weight components. Characteristics of this separation technique, known as micellar-enhanced ultrafiltration (MEUF), are that micelles bind specific compounds and subsequent ultrafiltration separates the surrounding aqueous phase from the micelles [70]. The pore size of the UF membrane must be chosen such, that the micelles are retained but the unbound components can pass the membrane freely. Alternatively, proteins such as BSA have been used in stead of micelles to obtain similar enan-tioselective aggregates [71]. 

For Yiv > YPv> where y v and Ypv are the surface tensions of liquid and protein, respectively, AFads increases with increasing ysv, predicting decreasing polymer adsorption. An example of this is phosphate buffer saline where y]v = 72.9 mJ/m2 and Ypv is usually between 65 and 70mJ/m2 for most proteins [5]. Therefore, supports for gel-permeation and affinity chromatography should be as hydrophilic as possible in order to minimize undesirable adsorption effects. 

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