Proteins solubilization

Fig. 2. Effect of changes ia system components on (—), emulsion stabifity and (---), protein solubilization ia an imitation milk where the protein and fat...

While the above discussion centered on the rate of disruption, the objective is usually to attain at least 90 percent release of the valuable protein from the cells. Cell disruption with protein solubilization is considered to be first order in amount of protein remaining [Currie et al., Biotechnol. Bioeng., 14, 725 (1972)] ... 

Product recovery from reversed micellar solutions can often be attained by simple backextraction, by contacting with an aqueous solution having salt concentration and pH that disfavors protein solubilization, but this is not always a reliable method. Addition of cosolvents such as ethyl acetate or alcohols can lead to a disruption of the micelles and expulsion of the protein species, but this may also lead to protein denaturation. These additives must be removed by distillation, e.g., to enable reconstitution of the micellar phase. Temperature increases can similarly lead to product release as a concentrated aqueous solution. Removal of the water from the reversed micelles by molecular sieves or silica gel has also been found to cause a precipitation of the protein from the organic phase. 

Subsequently, a mechanistic study demonstrated that protein solubilization in the organic phase occurred simultaneously with the adsorption of w/o-MEs by the solid phase [58]. Moreover, the results of this study supported the following proposed mechanism for SPE ... 

Lefkowitz, R. J., Haber, E., and O Hara, D. (1972). Identification of the cardiac beta-adrenergic receptor protein Solubilization and purification by affinity chromatography. Proc. Natl. Acad. Sci. USA 69, 2828-2832. 

To a small aliquot of the above buffer, add 2-mercaptoethanol to obtain a final concentration of 2-5 percent. Only 200 pi of this buffer typically is required to treat and reduce about 10-500 pg of protein. Solubilize the protein sample in this buffer. 

Subsequent to possible solubilization of membrane-bound proteins, solubilization must be verified. The criteria listed in Table 2 are relevant in assessing whether solubilization has been accomplished. To ascertain whether the solubilized protein has retained biological activity, membrane reconstitution (28) is attempted subsequent to detergent removal (24). Reconstitution is often visualized by electron microscopy employing either negative staining or freeze fracture. 

Summary of Criteria for Detergent Selection and Protein Solubilization... 

At concentrations well above the CMC, these effects are complicated by solubilization, but while it seems clear that protein solubilizing effectiveness and membrane activity correlate well, the structural basis for protein solubilization has not yet been clarified either. 

Effect of surfactant type and concentration An increase in surfactant concentration results in an increase in the number of micelles rather than any substantial change in size, and this enhances the capacity of the reverse micelle phase to solubilize proteins. Woll and Hatton [24] observed increasing protein solubilization in the reverse micelle phase with increasing surfactant concentration. In contrast, Jarudilokkul et al. [25] found that at low minimal concentrations (6-20 mmol dm AOT), reverse mieelles eould be highly seleetive in separating very similar proteins from... 

The information available in this polypeptide or these polyeptides is far from definitive. Controversy exists on size and molecular weight of the apo LDL monomers, their number, and chemical properties (Table 8). Also, the question of polypeptide heterogeneity has not been resolved this is not surprising in view of the difficulty of protein solubilization discussed earlier. At the time of this writing, apo LDL is the mostly poorly characterized protein of all serum lipoproteins. This apoprotein is... 

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-... 

Fig. 2. Schematic representation of mechanism of protein solubilization into reverse micellar phase from aqueous phase. (Reproduced from [45] with permission of Elsevier Science)...

Fig. 3a-c. Various methods of protein solubilization in reverse micelles. (Reproduced from [145] with permission of Wiley)... 

Blanch and coworkers [145] investigated in detail the solubilization properties of a-chymotrypsin and alcohol dehydrogenase (ADH) in RMs prepared by the above three techniques. Protein solubilization in RMs greatly depends on the method used for protein addition as well as on the size of the protein and of the RM. For the dry addition method protein solubilization is strongly dependent on micelle size whereas for the injection method it is less dependent. For smaller proteins like a-chymotrypsin (diameter of 44 A), maximum solubilization occurred when the micelle diameter was 50 - 60 A. For larger proteins like ADH... 

For the injection method, where a saturated protein solution is added directly to the surfactant-containing organic phase, the RMs are forced to form around the proteins already present in the solution. Thus, protein solubilization is not strongly dependent on micelle size in this technique. 

The phase transfer method of protein solubihzation is fundamentally different from the other two methods. In this method, there are two bulk phases (aqueous and organic) which are brought to equihbrium. Under certain conditions, the protein molecules are transferred from the aqueous phase to the surfactant-containing organic phase. Unhke the dry-addition and injection methods, it is difficult to obtain a value for the maximum solubihzation using the phase-transfer method. Moreover, since this method is mainly used for protein extraction, it is desirable to use aqueous phase protein concentrations consistent with those in a typical fermentation broth. For the phase-transfer method, pH of the aqueous phase, the size and isoelectric point of the protein, and the surfactant type were shown to have a significant effect on protein solubilization [145]. 

AOT/isooctane Chymotrypsin Presence of RMs is shown to be not a prerequisite for protein extraction. A new mechanism for protein solubilization has been proposed [299]... 

High pressure liquids and supercritical fluids were also served as solvent systems for the formation of RMs [342,343]. Franco et al. [344] implemented RMs in supercritical CO2 for protein solubilization. 

It is commonly agreed that enzymes are inactive in nearly anhydrous dimethyl sulfoxide (DMSO) [55] and that such inactivity might be a direct result of protein solubilization in the organic milieu, which causes deleterious changes in the proteins secondary and tertiary structures [56]. However, different authors have recently reported that some proteases, namely thermolysin (from Bacillus thermo-proteolyticus) and Proleather (from Bacillus subtilis), were still active in pure DMSO, despite the fact that proteins were indeed solubilized. The former enzyme cata-... 

Nielson, C. S. and Bjerrum, 0. J. 1977. Crossed immunoelectrophoresis of bovine milk fat globule membrane protein solubilized with non-ionic detergent. Biochim. Biophys. Acta 466, 496-509. 

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