Using sequence analysis you identify human proteins HKS1 and HKS2, which are mos

Question

Using sequence analysis you identify human proteins HKS1 and HKS2, which are most similar to yeast proteins YKS1 and YKS2. In cultures of normal human brain cells, you show that HKS1 and HKS2 localise to vesicles, like HKLP, when transfected with plasmids that express the proteins as GFP fusions. Page 4 You clone the HKS1, HKS2 and HKLP genes into bacterial expression systems that add a hexahistidine tag to their C-termini. Using Ni2+-beads, you purify each of the proteins and use them to raise antibodies in rabbits. You incubate human brain cell extracts with purified HKS2(His)6 and then add Ni2+- beads. You then spin these beads down and analyse the pellets by polyacrylamide gel electrophoresis. Finally you use a western blot to show that HKLP protein is present in the pellet pulled down by HKS2(His)6 bound to Ni2+-beads. When you repeat the experiment in vitro with purified recombinant protein, rather than cell extracts, you find that HKS2(His)6 does not pull down HKLP. You may assume that the hexahistine tag does not interfere with the interactions between proteins.

What is the most likely explanation for why HKS2 might not pull down HKLP in vitro but does so in cell extracts? How might you test this hypothesis with further “pull down” experiments?

Explanation / Answer

The most likely explanation for why HKS2 might not pulldown HKLP in vitro is due to the absence of HKS1 protein invitro experiment.

This suggest that the interaction between HKS2 and HKLP is indirect interaction mediated by HKS1 protein.

When we are incubating HKS2(His)6 with brain cell extracts, due to the presence of HKS1 in cell extracts there is an interaction and pulldown of HKLP.

In invitro experiment, HKS2 is incubated only with recombinant HKLP and thus due to the absence of HKS1 we couldnt pulldown HKLP.

We can test this hypothesis by incubating HKS2(His)6 with recombinant HKLP and HKS1 invitro. If we see pulldown of HKLP in HKS2(his)6 pulldowns, then we can say that HKS1 is mediatind the interaction between HKS2 and HKLP.

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