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Preparation, Analysis and Use of an Affinity Adsorbent 133
concentration of the amine and thiol groups, the total free ligand concentration
can be calculated. Ligand densities as high as 362 μmol/ml were observed for the
optimized immobilization protocol.
11. The materials and methods for bacterial cell transformation with the pM6 plasmid
and expression of the GST–ZnF are reported elsewhere (3,11).
12. An APV-2000 homogenizer unit (Invensys, Denmark) was used at a nominal
pumping rate of 11 l/h for minimum sample sizes of 100 ml.
13. Homogenization requires optimization for different cells and feed cell concentrations.
The method described in Subheading 3.5 was optimized via use of
SDS–PAGE gel analysis in order to obtain maximum yield of the GST–ZnF protein
(mg/ml) without degradation due to shear and/or an increase in temperature.
14. An Amersham Biosciences 5/5 column, 5 mm inside diameter, containing 1 ml of
adsorbent (5.1 cm bed height), was used and operated using an ÄKTA Explorer
(Amersham Biosciences). For this column, a flow rate of 0.2 ml/min equates to
approximately 60 cm/h.
15. Glutathione concentration considerations for GST–ZnF elution: A Biacore CM5
chip with covalently immobilized glutathione was used to determine the effect
of reduced glutathione concentration on the elution of GST–ZnF bound to the
glutathione ligand. After equilibration with PBS, a 25-μl sample containing 100
μg/ml of pure GST–ZnF was loaded onto the chip followed by washing and then
elution. Increasing concentrations of reduced glutathione in a solution of DI water
(pH 9) were used to determine the amount eluted, measured by the reduction in
response units (RU) from the start to the end of the elution. After each run, the chip
was regenerated and equilibrated. The results of the elution study are displayed
in Fig. 1. Significant increases in elution occurred as the reduced glutathione
concentration was increased from 0 up to 20 mM. From 20 mM up to 100 mM,
only minimal changes in elution were obtained (±8%). These variations were
within the experimental error (±27%). The data indicate that any further increase
in reduced glutathione concentration above 20 mM will not necessarily yield a
greater amount of eluted GST–ZnF. For an industrial scale operation, economic
issues would need to be considered as the ongoing costs of expensive eluting
agents (i.e., glutathione) is an important economic consideration and there is the
added processing issue of removing the eluting agent from the elution fractions.
It is therefore preferable to use the minimum amount of eluting agent whilst
maintaining optimal elution yields. The data presented in Fig. 1 supports the use
of 20 mM glutathione in the elution buffer.
16. pH considerations for GST–ZnF elution: Elution of GST–ZnF may be improved by
using an elution buffer pH where both the glutathione ligand and GST–ZnF have
the same charge (e.g., are both negative). The charge of a protein is determined
by its pI and the buffer pH, where the pI of a protein is the pH at which the
protein has an equal number of positive and negative charges. The number of net
negative charges on a protein increases with increasing pH above the pI (12). The
theoretical pI of GST–ZnF determined using the ExPASy ProtParam Tool (13,14)
is 8.96. An isoelectric focusing gel confirmed that the theoretical pI of the GST–