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In the blog post about the Tuberculosis Challenge ( https://fold.it/portal/node/2002586 ) free_radical says that the protein we were working on, LepB, is very hard to work with because it is bound in the membrane. Would it be possible to order a gene for it, leaving off the signal peptide and transmembrane anchor if it has them, maybe adding a HIS tag, and express it in E. coli the way you do with designed proteins? Maybe the portion we were working on could fold up and be extracted from the cytoplasm if the parts that normally migrate it into the membrane were left off.

Joined: 01/12/2015
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Hi Susume, The method that

Hi Susume,

The method that you mentioned is how I believe they are crystallizing the protein. They currently do not have the "tail" that sticks into the membrane on the protein, only the soluble portion. The way that they get this is how you mentioned, through creating/ordering the gene for the soluble portion. I am not sure if they are expressing the protein in e coli, but that would be a good choice. Sometimes proteins from other bacteria/organisms do not like to be expressed in e. coli, or when they are expressed, they do so in a very small amount. It could be that the protein is very toxic to its host.

Regardless of how it is expressed, part of the difficulty from expressing only the soluble portion is that you miss out on parts of the protein that may be "touching" and stabilized by the membrane. If these portions are not stabilized, they may unfold and cause poor diffraction patterns for the crystal. From what I have been told, it looks like they are still trying different crystallization methods for the protein soluble portion.

Part of the goal for modeling LepB is to find these erratic portions and insert mutations to stabilize them. We will be posting an extension (phase 3) to LepB puzzle in the upcoming weeks. Keep a look out for a new blog post!



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