Extraction of protein

Fluidised beds have been used previously for the industrial-scale recovery of the antibiotics streptomycin and novobiocin.30 However, more recently, considerable interest has been shown in the use of fluidised beds for the direct extraction of proteins from whole fermentation broths.31 In a packed bed, the adsorbent particles are packed within the contactor. The voidage, that is, the inter-particle space, is minimal and thus feedstock clarification is mandatory to avoid clogging of the bed. In a fluidised/expanded bed, the adsorbent bed is allowed to expand by irrigation with feedstock. Bed voidage is increased, allowing the passage of particulates in the feed. The diameters of the adsorbent beads are exaggerated for illustrative clarity. 

Jordan P, H Vilter (1991) Extraction of proteins from material rich in anionic mucilages partition and fractionation of vanadate-dependent bromoperoxidases from the brown algae Laminaria digitata and L. saccharina in aqueous polymer two-phase systems. Biochim Biophys Acta 1073 98-106. 

Extraction of protein from aqueous solution by surfactant-containing lipophilic organic solvent (phase transfer method, or equivalently, w/o-ME-based liquid-liquid extracting, LEE). 

To reduce the strong interactions occurring between protein and surfactant with AOT systems during LLE, anionic [8,32] and nonionic surfactants [30,33,142] and other interfacial additives [88,124,143] have been included. An improvement on the percent extraction of protein has also been reported for several of these cases [29,32,33,124,142,143]. 

TABLE 14.1 Extraction of Proteins from HeLa Cells Fixed in a 1% Agarose Plug... 

In summary, studies carried out with tissue surrogates25 highlight some of the problems that must be overcome before proteins extracted from FFPE tissues can be used for routine proteomic studies. First, these studies demonstrate that reversal of protein-formaldehyde adducts does not assure quantitative extraction of proteins from FFPE tissues or vice-versa. It may ultimately turn out that there is no one universal method that can accomplish both tasks, but that instead, each step will need to be optimized separately. Studies with tissue surrogates also suggest that failure to quantitatively extract the entire protein component from FFPE tissues may result in sampling bias due to the preferential extraction of certain proteins. This behavior may be linked to protein physical properties, such as the isoelectric point. The results of our... 

Recent studies have shown that HIAR is also effective for physically fixed materials, un-embedded and plastic-embedded specimens, and immunoelec-tron microscopy. Heating has also been shown to be useful for the extraction of proteins and nucleic acids from FFPE materials. The elucidation of the mechanisms described above should support the usefulness of such applications and lead to the establishment of standardized protocols for immunohis-tochemical staining. 

The objectives of the present study were to compare the processes of protein and starch concentration by dry air classification and wet alkali extraction of protein and starch from field pea and fababean. The yields, composition and functionality of the crude and refined products were compared. 

Table IX. Extractability of Protein from Aquatic Weeds3...

Counterion extraction Due to the relative slowness of back extraction based on the methods above, the back-extraction of proteins encapsulated in AOT reverse micelles was evaluated by adding a counterionic surfactant, either TOMAC or DTAB, to the reverse micelles [33]. This novel backward transfer method gave higher backward extraction yields compared to the conventional method. The back-extraction process with TOMAC was found to be 100 times faster than back-extraction with the conventional method, and as much as three times faster than forward extraction. The 1 1 complexes of AOT and TOMAC in the solvent phase could be efficiently removed using adsorption onto montmorillonite so that the organic solvent could be reused. 

The kinetics of back extraction are equally important to obtain a better understanding of the mechanism of solute transfer, and to determine the rate-limiting step for the process. Such information is crucial for the rational design of an extraction apparatus and, as discussed above, Jar-udilokkul et al. [33] showed that counterion extraction resulted in remarkable increases in the back-extraction of proteins. 

Automated robotic extraction of proteins from plant tissues... 

Brumback [28] has described a custom robotic system designed to automate the extraction of proteins from plant samples. Leaf or callus material (5—25 mg) is presented to the robot in microcentrifuge tubes, the system performs buffer dispensing, grinding, centrifugation, and pipetting unit operations, and a cleared supernatant is delivered in a 96-well microassay plate format for subsequent analysis. 

There are three commonly used methods [145] to incorporate enzymes in RMs (i) injection of a concentrated aqueous solution, (ii) addition of dry lyophilized protein to a reverse miceUar solution, and (iii) phase transfer between bulk aqueous and surfactant-containing organic phases. Figure 3 shows schematically the three enzyme solubiHzation methods. The injection and dry addition techniques are commonly used in biocatalytic appHcations, the latter being well suited to hydrophobic proteins [146]. The phase transfer technique is the basis for extraction of proteins from aqueous solutions. 

Efficient extraction of proteins has been reported with reverse micellar liquid membrane systems, where the pores of the membrane are filled with the reverse micellar phase and the enzyme is extracted from the aqueous phase on one side of membrane while the back extraction into a second aqueous phase takes place at the other side. By this, both the forward and back extractions can be performed using one membrane module [132,208]. Armstrong and Li [209] confirmed the general trends observed in phase transfer using a glass diffusion cell with a reverse micellar liquid membrane. Electrostatic interactions and surfactant concentration affected the protein transfer into the organic membrane and... 

Extraction of charged fusion gluco- [58] amylase (with short peptides) was carried out and maximal recovery (95%) was observed in CTAB system. Charged fusion process was shown to be advantageous and useful for extraction of proteins in RMs having small size... 

On the laboratory scale, RME for downstream processing of proteins is well established. To the best of our knowledge, there are no reports available on the larger-scale extraction of proteins using this technique, at levels above the 1-1 scale. The same is true with regard to continuous operation of RME, which was... 

Figure 4.16. Precipitation and extraction of proteins in stainiess steai stir-tanks in pharmaceutical scale preparations. (NiH Fredrick faciiity, with permission)...

The protocols for staining with amido black, Coomassie blue, Ponceau S, and AuroDye follow the suppliers recommendations. It should be noted that when staining PVDF membranes with Coomassie blue before N-terminal sequencing, omitting acetic acid from both the stain and destain solution is recommended to minimize potential extraction of protein from the membrane (Speicher, 1989). 

Before selecting a method to measure a specific aspect of protein functionality, one must decide on the complexity of the testing matrix. Researchers have used a single purified protein, a crude extract of proteins, a prototype food product, or an actual product to study protein functionality. For meat studies, formulated meat systems, ground muscle, myofibrillar proteins, salt-soluble proteins, actomyosin,... 

Once a crude extract of protein has been made, it is common to separate this mixture into different fractions by a precipitation step. The solubility of a protein reflects a delicate balance between different energetic interactions—both internally within the protein and between the protein and the surrounding solvent. Consequently, the choice of solvent can affect both the solubility and the structure of a protein. 

Hannah, M. J., Weiss, U., and Huttner, W. B. 1998. Differential extraction of proteins from paraformaldehyde-fixed cells Lessons from synaptophysin and other membrane proteins. Methods Enzymol. 26 170-181. 

T. Cunha and R. Aires-Barros, Large-scale extraction of proteins, Mol. Biotechnol. 

It also should offer promise for cell extractions, which, after all, are lipid/protein encapsulated mixtures of polar and nonpolar compounds. It would be interesting to see the effect of DMSO or DMF on the extraction of proteins. My guess is that protein might denature in 50% DMSO and precipitate so they could be filtered off or might renature and refold on lOx dilution in water and stay in solution. This might make an interesting research problem for recovering membrane-bound proteins. 

Similarly, the pulp and the seed of olives (Olea europaea) were extensively analyzed (48). Olives are constituted of a complex matrix for proteo-mics studies due to their lipidic nature and the large amount of interfering compounds from where the extraction of proteins is difficult (49).This fruit is used for the extraction of oils and as food ingredient in the Mediterranean diet. The olive pulp protein extraction was performed by freezing 5 g of olive... 

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