Purification factors

Another parameter used to characterise two-phase partitioning is the purification factor, defined as ... 

Stage Volume (ml) Total activity (IU) Total protein (mg) Specific activity (IU/mg) Purification factor Bound yield (%)... 

Ferric ion was immobilized on a Chelating Sepharose Fast Flow column preparatory to the separation of seven enkephalin-related phosphopep-tides.17 Non-phosphorylated peptides flowed through the column, and the bound fraction contained the product. The capacity of the column was found to be 23 pmol/mL by frontal elution analysis. Cupric ion was immobilized on Chelating Superose for the isolation of bovine serum albumin.18 Cupric ion was immobilized on a Pharmacia HiTrap column for the separation of Protein C from prothrombin, a separation that was used to model the subsequent apparently successful separation of Factor IX from prothrombin Factor IX activity of the eluate was, however, not checked.19 Imidazole was used as the displacement agent to recover p-galactosidase from unclarified homogenates injected onto a nickel-loaded IMAC column.20 Pretreatment with nucleases and cleaning in place between injections were required procedures. A sixfold purification factor was observed. 

Figure 5.2 The production and purification of tPAfrom the milk of transgenic goats. (WAP promoter murine whey acid promoter). The downstream processing procedure yielded in excess of an 8000-fold purification factor with an overall product yield of 25 per cent. The product was greater than 98 per cent pure, as judged by sodium dodecyl sulfate (SDS) electrophoresis...

The HbHnl is obtained from the leaves of the rubber tree plant and a crude extract is easily prepared by homogenisation of the frozen leaves, followed by centrifugation [38-40]. A 5-step purification procedure of this crude extract (with over a 100-fold purification factor) to yield a homogenous HbHnl has... 

Effects of GT-4 on Ileum. A constant infusion of GT 4 at nanogram levels produced exactly the same effects as previously described for the WSAP. Parallel assays with GT-4 and WSAP revealed that 2.8 ng/ml of GT-4 caused the same rate of decline of activity as 5.0 pg/ml of WSAP. These parallel assays of WSAP and GT-4 provided an efficient means for the estimation of a purification factor. Purification of the toxin was 1,785 fold. Using this value we projected an estimate for the LD50 of GT-4 as 0.62 pg/kg mouse. This value comes quite close to the calculated LD50 value reported for maitotoxin (0.17 pg/kg), isolated from toxicus from the Pacific. 

So that the purification of a given crystallization can be quantified, purification factors are defined with the following equation ... 

The notion of an ideal behavior also is defined here for those cases in which Pi is constant over a range of solution compositions, while variations with solution composition are said to characterize nonideal behavior. In the present studies values of purification factors are affected by the kinetics of the process. Accordingly, these quantities may not be true thermodynamic properties. 

Crystals Obtained by Acid Addition. Figure 4 shows the effect of initial solution composition on the impurity content of crystals obtained by acid addition. Clearly, this corresponds to the definition of an ideal system as presented above. These data show the order followed in impurity incorporation in the L-Ile crystals is L-Val > L-Leu > L-a-ABA, although there is only one data point on a-amino butyric acid. Also, the value of purification factors for all impurities is less than one. This means that purification by crystallization was indeed occurring. 

A series of runs was performed in which the acid addition rate was varied while holding the solution compositions and agitation constant Rvai, == 0.023, Rlcu s = 0.021, and 1000 RPM. The temperature was 25 C in all runs. Figure 5 shows that the purification factors were impacted by acid addition rate, and increased with the rate at which HCl was added to the system. The greatest effects are noticed below acid addition rates of about 5 g/min as the initial charge to the batch crystallizer was 150 g of solution, this corresponds to an addition rate of about 3.3% by mass per minute. 

Figure 5. Effect of Acid Addition Rate on L-Leu and L-Val Purification Factors in Recovered L-Ile HCl H20 Crystals...

In examining the effect of solution composition on the purity of recovered L-Ile crystals some unusual observations have been noted. Figure 7 shows data similar to that presented in Figure 4 for L-Ile HCMl20 with one major difference L-Valine is relatively unimportant as an impurity in comparison to L-Leu. Even though there is greater scatter in the data than was observed in the acid-addition experiments, purification factors for the two impurities are less than one and nearly constant. 

Since the recovery of neutral Lrlle may be performed after crystallization, redissolution and recrystallization, the concentrations of impurities in the solution were reduced by an order of magnitude in an additional series of experiments. Figure 8 shows the results. Once again L-VaJ is relatively unimportant and Puu appears constant, but note that the data do not go through the origin. Moreover, close examination shows that Puu > 1 which means that purification by crystallization has not occurred. Figure 9 shows that the purification factor for L-Leu is not constant and, therefore, the system is nonideal. 

Figure 9. Effect of Solution Composition on the Purification Factors for L-Leu and L-Val for Low-Impurity Solutions...

Purification step Volume activity [UmL J Protein concentration [mgmL j Specific activity lUmg ] Purification factor (fold)... 

Fraction Protein (mg) Total activity" Specific activity Yield (%) Purification factor... 

Fraction Protein (tug) Percent Recovery llutal Protein) Ptrwnt Rccuv n (Carrier Protein) Purification Factor... 

Purity is measured most conveniently by specific activity [U (mg protein)-1]. The ratio of the specific activity to the initial specific activity in the crude extract is called the purification factor. 

Purification protocols should always aim for brevity, to minimize complexity and cost. The goal of the protein purification procedure, besides a pure protein, is a purification table listing all the operations undertaken, with results on overall yield, specific activity, and purification factor. An assay for both protein function and protein concentration is required at every step. 

Purity is measured most conveniently by comparing specific activity [U [(mg protein)-1] at the processing stage in question to the specific activity of the pure protein. If the specific activity of the pure protein is not known, an estimate of the concentration of the target protein from gel electrophoresis can be substituted. The ratio of the specific activity at the processing step in question to the initial specific activity in the crude extract is called the purification factor. 

After homogenization, it is often advantageous to perform a salt precipitation, most commonly with ammonium sulfate. The purpose of this precipitation is the separation of cell debris and nucleic acids rather than the purification of the target protein from impurities. Whereas the purification factor of ammonium sulfate precipitation is usually around only 1.5 to 2, the separation of non-proteineous impurities and stabilization of the target protein in ammonium sulfate usually provide sufficient benefit to include this step in any purification protocol. 

Whether the goal is purification of a large-scale batch of protein for biocatalytic purposes or purification of an analytical amount to homogeneity for biochemical characterization of the protein, there is going to be a conflict between yield and purity. Despite the high salt levels required, ammonium sulfate precipitation remains one of the most effective methods for initial purification of proteins after fermentations, especially if an overexpressed protein has to be isolated even a purification factor of 2 increases purity significantly. Typical yields for this step should be expected to fall between 60 and 80%. 

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