Preparative Extractions

The following steps may be useful for a high degree of purification by affinity partitioning.

1. Pre-extraction in a system without ligand-PEG to remove proteins with relatively high partition coefficients. The target protein stays in the bottom phase by adjusting the choice of polymer concentration, salt and pH.

2. Affinity partitioning is carried out by changing the top phase for one containing ligand-PEG. The target protein will now be in the top phase.

3. Washing the top phase with bottom phase to remove co-extracted proteins.

4. 'Stripping' of protein from the affinity ligand by addition of highly concentrated phosphate solution (50%w/w) to the separated upper phase. This generates a PEG-salt two-phase system with PEG and ligand-PEG in the top phase and target protein in the salt-rich bottom phase. An alternative stripping procedure can be carried out by adding a new pure dextran phase to the recovered top phase and supplying the system with free ligand. In this case the target protein will be collected in the lower phase.

For each step the number of extractions and the most suitable volume ratios for yield and purity can be optimized. The procedure is summarized in Figure 8.

The yield in the top phase, YT, can be calculated from the K value of target protein and the volumes of top and bottom phase, VT and VR, respectively, using the following equation:

1 # Vb/(VtK) and the yield in the bottom phase, YB

A considerable concentration of the target protein, in addition to purification, can be achieved by choosing an extreme volume ratio with a small collecting phase.

An example of preparative extraction of an enzyme by applying the method given in Figure 8 is the purification of lactate dehydrogenase (LDH) using a PEG-bound textile dye. Crude extract of pig muscle,

Figure 8 Scheme for the purification of an enzyme (*) from contaminating proteins (■) by using four partitioning steps and PEG-dextran two-phase systems with PEG-bound ligand. This approach has been used for the purification of lactate dehydrogenase (LDH) from meat juice by affinity partitioning with Procion yellow HE-3G PEG. The inserted SDS-PAGE patterns of the original meat extract and the final product (obtained in the phosphate-rich phase) show the removal of contaminating proteins. Recovery of enzyme = 79%. System composition: 10%w/w dextran 500 and 7.1 %w/w PEG 8000 including 1% Procion yellow HE-3G PEG (of total PEG), 50 mM sodium phosphate buffer pH 7.9, and 25% w/w muscle extract. Temperature, 0°C. (Reprinted from Johansson G and Joelsson M (1986) Applied Biochemistry Biotechnology 13: 15-27, with permission from Elsevier Science.)

Figure 8 Scheme for the purification of an enzyme (*) from contaminating proteins (■) by using four partitioning steps and PEG-dextran two-phase systems with PEG-bound ligand. This approach has been used for the purification of lactate dehydrogenase (LDH) from meat juice by affinity partitioning with Procion yellow HE-3G PEG. The inserted SDS-PAGE patterns of the original meat extract and the final product (obtained in the phosphate-rich phase) show the removal of contaminating proteins. Recovery of enzyme = 79%. System composition: 10%w/w dextran 500 and 7.1 %w/w PEG 8000 including 1% Procion yellow HE-3G PEG (of total PEG), 50 mM sodium phosphate buffer pH 7.9, and 25% w/w muscle extract. Temperature, 0°C. (Reprinted from Johansson G and Joelsson M (1986) Applied Biochemistry Biotechnology 13: 15-27, with permission from Elsevier Science.)

cleared by centrifugation, is mixed with PEG, dextran and Procion yellow HE-3G PEG. After the first partitioning the top phase is washed twice with pure lower phases and then it is mixed with a 50% w/w salt solution (25% NaH2PO4 + 25% Na2HPO4• H2O). The protein content of the final product in the salt-rich phase compared with that of the initial extract is demonstrated by the polypeptide pattern in sodium dodecyl sulfate-polyacryl amide gel elec-trophoresis (SDS-PAGE) shown in Figure 8. The

L-PEG (and PEG) recovered in the final top phase is ^95% of the initially introduced amount.

Purification of PFK in combination with a precipitation step with PEG before the affinity partitioning step greatly reduces the original volume of enzyme solution. The extraction included both pre-extraction and washing steps. The final polishing of the enzyme was made by ion exchanger and desalting with gel chromatography. The results can be seen in Table 3.

Table 3 Purification of phosphofructokinase from 1 kg (wet weight) bakers' yeast

Purification step

Volume

Total

Total

Specific

Purification

Yield

Proteolytic

(ml)

activity

protein

activity

factor

(%)

activitya

(U)

(mg)

(U/mg)

(%)

Homogenate

1370

5400

13170

0.41

1

100

100

Fractional precipitation

120

4810

1836

2.62

6.4

89

18

with PEG

Affinity partitioning

120

3610

153

23.6

58

67

0.9

DEAE-cellulose

40

2520

63

40

98

47

0.4

treatment

Gel filtration

4

1625

28

58

142

30

0.05

aIn the presence of the protease inhibitor phenylmethylsulfonyl fluoride.

aIn the presence of the protease inhibitor phenylmethylsulfonyl fluoride.

Figure 9 Affinity extraction into the top phase, by using increasing amount of PEG-bound ligand, calculated for an enzyme with the mole mass 100 000 g mor1, containing two binding sites for the ligand, and with KP = 0.01. The value for the partition coefficient, KL, of the ligand is 100. The association constant, K for each site is 10~6 M~1 (O, □) or 10~4 IVT1 ( #). The concentration of enzyme: 10 (O), 100 (□, #) and 500 g L~1 (A).

Figure 9 Affinity extraction into the top phase, by using increasing amount of PEG-bound ligand, calculated for an enzyme with the mole mass 100 000 g mor1, containing two binding sites for the ligand, and with KP = 0.01. The value for the partition coefficient, KL, of the ligand is 100. The association constant, K for each site is 10~6 M~1 (O, □) or 10~4 IVT1 ( #). The concentration of enzyme: 10 (O), 100 (□, #) and 500 g L~1 (A).

The effectiveness of affinity partitioning depends on the binding strength between ligand and protein. Good extraction is obtained with association constants of 104M_1 or more (Figure 9). The capacity, based on the amount of ligand in the system, is in the range of several hundred grams of protein per kilogram of system. Affinity extractions with 150 g of protein per kilogram of system have been carried out, and in these cases the two-phase systems strongly change the phase volume ratio while the bulk protein acts as a phase-forming component. In systems with high protein concentration the amount of dextran can be reduced or even excluded.

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