Chemistry Reference
In-Depth Information
the resin and thus to compare enzymatic activity of the target prior to and after immobiliz-
ation, there was an equivalent presence of resin in both cases. Results showed an efficient
enzymatic activity post-immobilization. Considering the imprecision in determining the
amount of immobilized enzyme, the rate of the reaction of immobilized enzyme (3 M
Ubiquinone-5/M DsbB s
−
1
) was close to that of the enzyme in the presence of, but not
immobilized on, resin (4 M Ubiquinone-5/M DsbB s
−
1
) (Figure 6.4).
No DsbB
2.0
Solution
1.5
Immobilized
1.0
0.5
No Quinone
0
0
20
40
60
80
100
Time (sec)
Figure 6.4
The target immobilized to the resin shows similar enzymatic activity of to the
target in the presence of, but not immobilized to, the resin.
Naturally, more complex strategies can be envisioned and may prove necessary for
membrane proteins that are less robust than those used so far. One interesting strategy
immobilizes protein first, followed by subsequent reconstitution into a synthetic lipid envir-
onment.
[
42
]
As with soluble proteins, active site blockers may be necessary in cases where
illicit immobilization of lysine side-chains in close proximity to the binding site may occur
and thereby inhibit protein function. Various native or synthetic lipid assemblies have been
extended to en
c
ompass the use of high-affinity immobilization reagents such as biotin and
streptavidin,
[
43 46
]
antibodies
[
47 49
]
or metal affinity
[
50, 51
]
in order to immobilize the protein
in more oriented manners. Therefore, as with soluble proteins, these approaches should
also be compatible with TINS.
As a first step along the road to enabling TINS ligand screening for a truly broad range
of membrane targets, we have begun to immobilize GPCRs in native membrane frag-
ments (Früh
et al
., in preparation). In this experiment, the idea was to use standard, stable
animal cell expression systems such as CHO or HeLa cells as a source of material. In this
way, all membrane proteins that can be recombinantly expressed in these simple systems
could potentially be used in fragment screening campaigns. Thus far we have succeeded
in immobilizing membrane fragments produced by pottering (gentle disruption of animal
cells) of post-centrifugation membrane preparations. We have applied the procedure to
both histamine receptors and adenosine receptors and, in both cases, the pharmacology
of immobilized receptors was similar to that of nonimmobilized receptors. The efficiency
of immobilization is reasonable, with approximately 20% of total receptors functionally
