I am trying to create some constructs of a certain protein deleting well defined

Question

I am trying to create some constructs of a certain protein deleting well defined domains (at either terminus) to determine interaction regions with other proteins etc., 3 constructs with varying start/end sites have ended up being aggregated in E coli (while the full length protein expresses reasonably well). My question is what considerations one should use to determine start/end sites to maximize chances of getting soluble, purifiable protein. The criterion I used were:

From personal experience with an earlier construct, this can turn out to be an idiosyncratic exercise but I was wondering if I was missing any crucial parameters.

Explanation / Answer

Did you identify structural domains in the protein using Pfam or some other domain detecting peptide search?

It's pretty useful to cut your constructs at the boundaries of structurally independent domins in the protein if you know where they are. Hydrophobicity plots might lead you to cut right in the middle of a protein fold, only exascerbating inclusion body formation/protein precipitation.

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