Key words: potato proteins, patatin, protease inhibitors, solubility, structure, pH, temperature, ethanol, ionic strength, phenolic compounds, foams, emulsions In potato starch manufacture an aqueous byproduct remains that is called potato fruit juice (PFJ). On a dry matter basis PFJ contains about 20-25 % protein and amino acids, 15 % sugars, 20 % minerals, 14 % organic acids and other components, such as phenolic compounds. Potato protein has a relatively high nutritional quality, comparable to that of whole egg, and it therefore has high potential for utilization in food applications. Protein recovery from industrial PFJ is presently achieved through heat coagulation by steam injection after pH adjustment. This method is very efficient in removing protein from solution. However, it leads to protein precipitates that exhibit a poor solubility, which hampers potential food applications. An economic method to efficiently recover soluble potato protein would considerably increase its possibilities for use in food and add to its commercial value. Therefore, the important question resulting in this study was: can potato proteins be recovered from PFJ in such a way that they retain their functional properties, most importantly their solubility? This recovery method should be applicable at a large scale and result in a high yield. Potato protein recovery was expected to be complicated by the presence of and the interactions with non-protein components in PFJ. The objective in this study was to examine how extrinsic factors like pH, ionic strength and temperature would influence the structure of potato proteins, this in relation to the functionality of the proteins in making and stabilizing foams and emulsions. Three groups of potato proteins can be distinguished in PFJ. Patatin, the major potato tuber protein, comprises 38 % of the protein in PFJ from cultivar Elkana . The protease inhibitors make up about 50 % and other proteins up to 12 % of total protein in PFJ from cultivar Elkana . In Chapter 2 the effects of pH and various additives on the precipitation and (re)solubility at pH 7 of potato proteins from industrial PFJ are studied. Addition of various strong and weak acids caused the same extent of protein precipitation, which comprised at the most 60 % of total protein at pH 3. The use of weak acids, however, resulted in an increase in the resolubility of the precipitates at pH 7, as compared to strong acids. At pH 5 addition of FeCl 3 or ZnCl 2 increased both precipitation and resolubility. The largest increase in precipitation and resolubility was achieved by using organic solvents, resulting in a maximum precipitation (pH 5) of 91 % of total protein and a maximum resolubility of 91 % of precipitated protein. The results described in Chapter 2 lead to the hypothesis that precipitation and resolubilization of potato proteins from PFJ is not so much determined by their isoelectric pH but by their interactions with low molecular weight components. In Chapter 3 it was shown, using DSC and both far-UV and near-UV CD spectroscopy, that potato proteins unfold between 55°C and 75°C. Increasing the ionic strength from 15 to 200 mM generally caused an increase in denaturation temperature. It was concluded that the dimeric protein patatin unfolds either in its monomeric state or that its monomers are loosely associated and unfold independently. Thermal unfolding of the protease inhibitors was correlated with a decrease in protease inhibitor activities and resulted in an ionic strength dependent loss of protein solubility. Potato proteins were best soluble at neutral and strongly acidic pH. At mildly acidic pH the overall potato protein solubility was dependent on ionic strength and the presence of unfolded patatin. In Chapter 4 a protein isolate with a high solubility at neutral pH prepared from industrial PFJ by precipitation at pH 5 in the presence of ethanol is described. The effects of ethanol itself and the effects of its presenc

Mixed micelles as proliposomes for the solubilization of teniposide. The aqueous solubility of teniposide in detergent and phospholipid mixed micelles was investigated as functions of the detergents and lipids composing the mixed micelles, the molar ratio of detergent to phospholipid, and the total lipid concentration of the system. The polarity, the charge of the phospholipid, and its saturation affected the solubilization potential of the micelles. Physical chemical factors such as the pH, ionic strength, and temperature of the dispersion medium also altered the solubilization capacity of the system. The results are explained by the changes occurring in the critical micelle concentration and packing arrangements of the aggregates. The desired solubility of teniposide can be achieved by adjusting the studied parameters to the optimum values. Teniposide-containing mixed micelles were spontaneously converted to drug-containing vesicles upon aqueous dilution; therefore, the precipitation of the drug was totally eliminated. In conclusion, mixed micelles as proliposomes can be a suitable drug carrier system for insoluble compounds such as teniposide.

Recombinant proteins play many important roles in development of biological reagents and biopharmaceuticals. Here, we will mainly review refolding of recombinant proteins when expressed in inclusion bodies, although strategies to enhance soluble expression are described as an alternative to refolding inclusion bodies. These strategies include, but not limited to, adding chemical chaperones in cell culture media, modifying cell lysis buffer and using solubility-enhacing fusion tags. Another solubility enhancement was to generate lipid complex for membrane proteins that form insoluble proteins without lipid. Among various solubilization and refolding technologies, those using denaturant, alkaline pH and pressure are also desribed, while we focus on solubilization and refolding using detergents, which are effective and cost-friendly. Sodium dodecylsulfate, lauroyl-glutamate, sarkosyl and cetyltrimethylammonium have been extensively used, as summarized in this review. Slow or step-wise removal of denaturants or ionic detergents used to solubilize appears to play a critical role in successful refolding by maintaining the solubility of proteins during refolding. In alkaline refolding, slow pH adjustment also helps maintain protein solubility. In pressure refolding, small amount of guanidine hydrochloride assisted refolding.

Ionic detergents like sodium dodecyl sulfate (SDS) and cationic detergents like ethyl trimethyl ammonium bromide are denaturing (will disrupt protein A lysis buffer is a buffer solution used for the purpose of breaking open cells for use in molecular biology experiments that analyze the labile macromolecules of the cells (e.g. western blot for protein, or for DNA extraction). Most lysis buffers contain buffering salts (e.g. Tris-HCl) and ionic salts (e.g. NaCl) to regulate the pH and osmolarity of the lysate. Sometimes detergents (such as Trito A lysis buffer is a buffer solution used for the purpose of breaking open cells for use in molecular biology experiments that analyze the labile macromolecules of the cells (e.g. western blot for protein, or for DNA extraction). Most lysis buffers contain buffering salts (e.g. Tris-HCl) and ionic salts (e.g. NaCl) to regulate the pH and osmolarity of the lysate. Sometimes detergents (such as Triton X-100 or SDS) are added to break up membrane structures. For lysis buffers targeted at protein extraction, protease inhibitors are often included, and in difficult cases may be almost required. Lysis buffers can be used on both animal and plant tissue cells. Detergents are organic amphipathic (with hydrophobic tail and a hydrophilic head) surfactants. They are used to separate membrane proteins from membrane because the hydrophobic part of detergent can surround biological membranes and thus isolate membrane proteins from membranes. Although detergents are widely used and have similar functions, the physical and chemical properties of detergents of interest must be considered in light of the goals of an experiment. Detergents are often categorized as nonionic, anionic, cationic, or zwitterionic, based on their hydrophilic head group feature. Nonionic detergents like Triton X-100 and zwitterionic detergents like CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate) are nondenaturing (will not disrupt protein functions). Ionic detergents like sodium dodecyl sulfate (SDS) and cationic detergents like ethyl trimethyl ammonium bromide are denaturing (will disrupt protein functions). Detergents are a major ingredient that determines the lysis strength of a given lysis buffer. One common issue faced by many cell lysis buffers is the disruption of protein structures during the lysis process, partially caused by use of detergents. Detergents often prevent the restoration of native conditions necessary for proper protein folding. For the longest time, after a detergent-based cell lysis, a buffer exchange and/or dialysis had to be performed to remove the detergent among other hindering compounds to restore native conditions. To overcome this a solution has emerged in… T…