Precipitation protein recovery

Protein recovery via disruption has also been achieved by adsorbing water from the w/o-ME solution, which causes protein to precipitate out of solution. Methods of water removal include adsorption using silica gel [73,151], molecular sieves [152], or salt crystals [58,163], or formation of clanthrate hydrates [154]. In most of the cases reported, the released protein appeared as a solid phase that, importantly, was virtually surfactant-free. In contrast to the dilution technique, it appears that dehydration more successfully released biomolecules that are hydrophilic rather than hydrophobic. 

Relative extraction efficiencies of polar polymeric neutral, cation, and anion exchange sorbents (HLB, MCX, and MAX) for 11 beta antagonists and 6 beta agonists in human whole blood were probed.109 Initial characterization of MCX and MAX for acidic and basic load conditions, respectively, showed that both the agonists and antagonists were well retained on MCX, while they were recovered from MAX in the wash with either methanol or 2% ammonia in methanol (see Table 1.6). Blood samples were treated with ethanol containing 10% zinc sulfate to precipitate proteins and the supernatants loaded in 2% aqueous ammonium hydroxide onto the sorbents. After a 30% methanol and 2% aqueous ammonia wash, the analytes were eluted with methanol (HLB), 2% ammonia in methanol (MCX), or 2% formic acid in methanol (MAX). The best recoveries were observed with MCX under aqueous conditions or blood supernatant (after protein precipitation) spiked sample load conditions (see Table 1.7). Ion suppression studies by post-column infusion showed no suppression for propranolol and terbutaline with MCX, while HLB and MAX exhibited suppression (see Figure 1.6). 

The study concluded that Once wash steps are optimized, samples prepared by solid phase extraction are cleaner than those prepared by protein precipitation. Samples prepared by extraction with a Multi-SPE plate resulted in lower LOQs than samples prepared by solvent precipitation. Drug recoveries were acceptable (>80%) for both the SPE and the solvent precipitation methods. Well-to-well reproducibility of samples was slightly better with extraction with a Multi-SPE plate. Evaporation and reconstitution, while more time-consuming, yield better chromatographic performance, allow analysis of lower concentration samples, and require optimization for good analyte recovery. 

Thermal denaturation, recovery of precipitated protein by filtration/ centrifugation and spray-drying, to yield lactalbumin which has very low solubility and limited functionality. 

Ninhydrin forms fluorophors of high intensity with guanidino compounds in alkaline media. Dihydrostreptomycin, which has two guanidino groups, yields similar fluorophors. Milk sample was treated with TCA, to precipitate proteins, and extracted with dichloromethane and NaOH, and the supernatant cleanup was performed using a Cl8 SPE column. The analyte was eluted with formic acid in MeOH. The postcolumn derivatization was performed at 80°C. The recovery from all procedures varied from 82.6% to 82.8% (only for two concentration levels), with RSD of 0.7-1.2%. This method can also be used for the determination of STR in milk (112). 

Once the crude cellulase solution was obtained, it was concentrated and at the same time separated from most of the salts (left from the fermentation) in the enzyme solution. The steps which accomplished this were (1) addition of ammonium sulfate (75% saturation) to precipitate protein (2) recovery of the protein as a pellet by centrifugation at 12,000 rpm for 15 min (3) redissolution of the protein in 0.1M sodium phos-phate-0.2mM EDTA buffer (pH 6.8) (4) desalting on a 1.5 X 45 cm Sephadex G-25 column and (5) lyophilization to obtain concentrated enzyme followed by a final ammonium sulfate precipitation to obtain precipitated enzyme. 

Goitrous hypothyroidism has rarely been described in patients taking ethionamide (393), with recovery after withdrawal (394). Ethionamide inhibits both the uptake of iodine and its incorporation into trichloroacetic acid-precipitable protein (395). 

A number of applications are given in this chapter that detail the capture and recovery of intracellular proteins including recombinant proteins. The initial protein recovery steps, regardless of the source, are usually associated with large volumes and crude solutions, requiring removal of particles and reduction of volume before their purification can take place. Centrifugation, filtration, precipitation, solvent extraction, and batch adsorption are common unit operations involved in the preliminary steps of protein recovery. Expanded-bed adsorption, as described here, is an approach for the initial protein recovery that eliminates the need for clarification and volume reduction. In this process, a crude starting solution is pumped directly onto an... 

These results demonstrated the feasibility of EDBM for whey protein separation and the influence of the initial protein concentration on the purity and yield of the separated fraction. At 5% WPI initial concentration, this technology allowed the separation of 98% pure (3-lg fraction with a 44% recovery yield, while at 10% WPI initial concentration a (3-lg-enriched fraction was produced containing 97.3% of (3-lg and 2.7% a-la, for a 98% total protein purity. The 10% protein concentration seems to be the best level for electrodialytic parameters and protein recovery. Furthermore, EDBM of a 10% WPI solution, by precipitation of 53.4% of the (3-lg, allowed the production of an a-la-enriched fraction in the supernatant. Since the best pH to precipitate (3-lg was demonstrated to be pH 4.65 [21], and that the protein yield increases with an increase in initial protein concentration in the solution, it was expected that electroacidilication of a 20% WPI solution to pH 4.65 would allow the highest precipitation yield. However, the limiting factor of such a process at 20% was the low conductivity of the protein solution at pH 5.0. 

Protein precipitation as a sample pretreatment method is very popular in quantitative bioanalysis, because it is a very fast and almost generic approach. First, the protein precipitation additive is added. After mixing and centrifugation, the supernatant can be directly injected into the LC-MS system. Typical additives are trichloroacetic acid (TCA), zinc sulfate, acetonitrile, ethanol, or methanol. The use of zinc sulfate in LC-MS requires a divert valve to avoid excessive somce contamination. TCA might result in significant ion suppression. In some cases, poor analyte recovery is observed, probably due to inclusion of analytes in the precipitating proteins. 

The product is often purified further after the recovery phase, particularly when the desired product is a protein of pharmaceutical interest. Precipitation is a widely used method that can be induced by the addition of salts, organic solvents, or heats. It can both purify and concentrate a particular protein fraction, and is frequently accomplished by the addition of salts for a salting out effect. The addition of a salt precipitates proteins because increasing salt concentrations reduce the solubility of a protein in a solution. Even though precipitation is an effective and relatively inexpensive method, it is also a fairly crude step and is often followed by chromatographic separations. ... 

Interactions between protein and polyelectrolytes have been used to fractionate protein solutions (1-3), recover whey proteins (1, 3-6) and isolate serum glycoproteins (7) and recA protein (8). If the potential for protein recovery and purification from aqueous solution by precipitation with polyelectrolytes is to be fully exploited, several factors must be evaluated. The efficacy of the protein precipitation as well as the characteristics of the resulting precipitates depend on several variables. 

Earlier work (9) has shown that the size of the precipitate, but not the protein recovery, depends on the method of addition of the polymer to the protein solution. Mixing conditions in the precipitation vessel also affect the precipitate size (lOh The solubility of the protein-polyelectrolyte complex depends strongly on the solution conditions—pH, ionic strength, polymer dosage level, and the nature of the protein and polyelectrolyte. These factors are discussed below ... 

Several conclusions can be drawn about the effect of polymer dosage, pH and ionic strength on protein recovery and fractionation by precipitation with CMC ... 

Increased ionic strength levels serve to increase the polymer dosage requirement, reduce the maximum precipitation possible, and reduce the effect of precipitation pH on protein recovery. 

A review of efforts to experimentally characterize and model the phenomena important in protein precipitation shows that, despite successes, continued work is necessary to produce accurate mechanistic descriptions of this method of protein recovery. 

Cheese Whey Protein Recovery. Perhaps the best publicized application for UF is in cheese whey processing. "Cheese whey" is the supernatant liquid produced in the cheese making process after precipitation of casein from milk. There are two types of whey "sweet" whey (minimum pH of 5.6) results when rennet-type enzymes are used to coagulate the casein to form Gouda and Cheddar cheeses ... 

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