Calcium-Binding Protein Protocols Calcium-Binding Protein Protocols

332 Kobayashi
0.1% TFA at a flow rate of 1 mL/min (at 13.3%/min for min). Then apply a
shallow acetonitrile gradient: 40–64% acetonitrile at 0.34%/min.
4. Collect UV absorbing material using a peak-actuated fraction collector.
5. Lyophilize samples and analyze for purity on Tricine/SDS/PAGE (15). To identify
the protein, subject purified sample to TOF-mass and/or protein sequencing
analysis.
3.8. Analysis of Ca2+ -Dependent Interaction of S-100 Family
Proteins and their Mutant Proteins with Immobilized Drugs
EF-hand Ca2+ -binding proteins, such as calmodulin and S-100 protein family
interact with calmodulin antagonists in a Ca2+ -dependent manner. These
calmodulin antagonists also inhibit the Ca2+ -dependent activation of enzymes
by calmodulin and therefore are useful probes of the relationships between
structure and function in calmodulin and S-100 protein family. Recently, a new
class of selective S-100 interacting agents, such as Cromolyn, Amlexanox, and
Tranilast (antiallergic drugs), are reported. In this Subheading, we describe the
advantage of affinity chromatographic analysis for Ca2+ -dependent drug-protein
interaction (see Note 2).
3.8.1. Preparation of Standardized Drug-Affinity Columns
1. Pour the slurry of a drug-immobilized matrix (1 mL of bed volume) into Polyprep-
Column (Bio-Rad) and equilibrate the column with 10 mL of Buffer B.
2. Apply the protein sample (150 µg) in a small volume of Buffer A and elute the
column with 10 mL of the same buffer. Collect each 1 mL of eluate.
3. Elute the bound protein successively with 10 mL of Buffer C and 10 mL of Buffer
D. Collect each 1 mL of eluate.
4. To identify the protein, subject each fraction to Tricine/SDS/PAGE (15).
3.8.2. Example 1: Affinity of Recombinant
S100A12 and S100A13 to Amlexanox (13,14)
The affinity of recombinant S-100A12 and S-100A13 to the anti-allergic
drug, Amlexanox, was examined. The recombinant proteins expressed from
the bovine lung cDNA were applied to the Amlexanox-AF amino Toyopearl
column (1 mL) with the Ca 2+ containing buffer (Buffer B). After washing the
column with the same buffer, the bound protein was eluted with 2 mM EGTA
(Buffer C). As shown in Fig. 4, the elution pattern and 12% Tricine/SDS/PAGE
of recombinant S-100A12 indicated that it bound to Amlexanox in the presence
of Ca 2+ , and dissociated from the drug by removing Ca 2+ from the protein.
The recombinant S-100A13 was also examined in a similar manner and it was
found that this protein also bound to the drug in a Ca 2+ -dependent manner,
although a large part of S-10A13 passed through the column.