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Q51 - Phage-Display Screen for Protein Kinase Inhibitors

Theoretical B Real exam question - full text reproduced under IBO's CC BY-NC-SA 4.0 license
Recombinant DNA & Biotechnology Techniques

A scientist prepared 3 essential components for high-throughput screens of protein kinase inhibitors. First, individual protein kinase genes are fused to the major capsid (head) gene of T7 phage. When expressed in bacteria, the fusion proteins are assembled into the phage capsid, with the kinases displayed on the outer surface. Second, an analog of ATP, which can bind to the ATP-binding pocket of the kinases, is attached to magnetic beads. Third, a bank of test compounds is prepared.

Figure Q.51 Screening potential inhibitors of protein kinases

To measure the ability of a test compound to bind a kinase, phage displaying a specific kinase is mixed with the magnetic beads in several wells of a 96-well plate. Then the test compound is added to individual wells over a range of different concentrations. The mixtures are incubated with gentle shaking for 1 hour at 25°C, the beads are pulled to the bottom with a strong magnet, and all the free (unbound) components are washed away. Finally, the remaining, attached phage are dissociated from the beads using an excess of the same ATP analog that is attached to the beads, and counted by measuring the number of plaques they form on a bacterial lawn on a Petri dish (Fig.Q.51).

Figure 1. Figure 1.

Using the information and data, determine which of the statements are true or which are false.

A. When the binding process reaches equilibrium, all potential inhibitor molecules will be bound to the kinase.
B. Test compounds that show high inhibition in this assay must bind the ATP-binding cleft of the kinase.
C. Small differences in evolutionary conserved ATP binding sites on kinases allow targeting specific kinases.
D. A strongly binding test compound will yield a low count in the plaque assay.
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Question reproduced from IBO 2016, Theoretical Paper B, licensed under CC BY-NC-SA 4.0 - attributed to the International Biology Olympiad. Open the full exam PDF · Community solutions (unofficial)