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134 Forde
250
Elution in response units (RU)
200
150
100
50
0
0 20 40 60 80 100
GHS Concentration (mM)
Fig. 1. Elution profile of GST–ZnF eluted from glutathione ligand immobilized
onto a Biacore CM5 chip. A 25-μl sample containing 100 μg/ml of pure GST–ZnF
was loaded onto the chip followed by elution. The amount of GST–ZnF eluted was
determined by measuring the change in RU before and after loading of the elution
buffer.
ZnF is approximately correct (data not shown). Studies of the effect of pH on the
elution of GST–ZnF from an affinity adsorbent were performed. The elution pH
was varied whilst maintaining constant glutathione (20 mM) and Tris–HCl (100
mM) concentrations. Clarified lysate containing GST–ZnF (1 ml, 1.34 mg/ml)
was bound to 0.1 ml of affinity adsorbent (5-min incubation) and eluted using
1.50 ml of elution buffer (5-min incubation). The amount of total protein eluted
was measured by a bicinchoninic acid protein assay and the percentage of total
protein that was GST–ZnF determined by a densitometry study of an SDS–PAGE
gel, shown in Fig. 2.
The amounts of GST–ZnF eluted were 0.41 mg/ml adsorbent for pH 8, 0.73 mg/ml
adsorbent for pH 9 and 0.90 mg/ml adsorbent for pH 10. Amersham Biosciences
(15) recommends an elution buffer of pH 8 and a maximum elution buffer pH of
9. At pH levels above 9, GST fusion proteins will be denatured (16), jeopardizing
the function of the proteins (i.e., ability to bind to affinity ligands or recognition
sequences). The pI of recombinant GST expressed by E. coli is 6.52 (17). An
elution pH of 8 is sufficient for the successful elution of GST from glutathione
ligands. However, the pI of the GST–ZnF fusion protein (8.96) is higher than
that of GST. Using an elution buffer of pH 9 ensures that the pH is above the pI
for both the ligand and target protein whilst the pH is at a level which will not
denature the fusion protein.