General protein purification

In order to be exploitable for extraction and purification of proteins/enzymes, RMs should exhibit two characteristic features. First, they should be capable of solubilizing proteins selectively. This protein uptake is referred to as forward extraction. Second, they should be able to release these proteins into aqueous phase so that a quantitative recovery of the purified protein can be obtained, which is referred to as back extraction. A schematic representation of protein solubilization in RMs from aqueous phase is shown in Fig. 2. In a number of recent publications, extraction and purification of proteins (both forward and back extraction) has been demonstrated using various reverse micellar systems [44,46-48]. In Table 2, exclusively various enzymes/proteins that are extracted using RMs as well as the stability and conformational studies of various enzymes in RMs are summarized. The studies revealed that the extraction process is generally controlled by various factors such as concentration and type of surfactant, pH and ionic strength of the aqueous phase, concentration and type of CO-surfactants, salts, charge of the protein, temperature, water content, size and shape of reverse micelles, etc. By manipulating these parameters selective sepa-... 

The various steps involved, in general, which need to be considered for the development of a process for the extraction and purification of proteins by RME are ... 

The extraction and purification of proteins from organisms or biological tissue can be a laborious and expensive process, and often represents the principal reason why vaccines and other therapeutic agents reach costs that become unattainable for many. Downstream processing also can be a major obstacle with respect to cost for large-scale protein manufacturing in plants. However, purification from plant tissues, while still costly, is in general less expensive than purification from their mammalian and bacterial counterparts. Indeed, some plant-derived biopharmaceuticals, such as topically applied monoclonal antibodies, may require only partial purification and thus be even less intensive in terms of labor and cost. 

A number of other precipitating agents have been used in the purification of proteins. In some instances, the use of sodium sulfate can result in a purer antibody preparation, but generally it does not offer advantages over ammonium sulphate. Caprylic (octanoic) acid, however, offers a different approach and also has a long history of use (5). Conditions can be created where this short chain fatty acid will effectively precipitate the majority of serum proteins with the exception of the immunoglobulins. 

Many different proteins exist in a single cell. A detailed study of the properties of any one protein requires a homogeneous sample consisting of only one kind of molecule. The separation and isolation, or purification, of proteins constitutes an essential first step to further experimentation. In general, separation techniques focus on size, charge, and polarity—the sources of differences between molecules. Many techniques are performed to eliminate contaminants and to arrive at a pure sample of the protein of interest. As the purification steps are followed, we make a table of the recovery and purity of the protein to gauge our success. Table 5.1 shows a typical purification for an enzyme. The percent recovery column tracks how much of the protein of interest has been retained at each step. This number usually drops steadily during the purification, and we hope that by the time the protein is pure, sufficient product will be left for study and characterization. The specific activity column compares the purity of the protein at each step, and this value should go up if the purification is successful. 

The nse of aptamers for the separation, pnriflcation, and quantification of analytes in chromatography, electrochromatography, and capillary electrophoresis techniqnes in general is described in detail and with examples by Ravelet et al. (2006). DNA and RNA aptamers have been nsed for the separation and purification of proteins and the separation of small molecnles and enantiomers. In capillary electrophoresis, aptamers are nsed primarily for the separation of species and the characterization of affinity interactions. Ravelet et al. (2006) state the hnge potential of these molecular tools in the separation science field. However, for big separation units, a large amonnt of aptamer is necessary, making it more expensive than other separation materials. Therefore, the nse of aptamers for separation units is limited primarily to miniatnrized systems. 

Establishment of general methods for purification of proteins that bind riboflavin purification of 221 riboflavin carrier protein, flavokinase and flavodoxin... 

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