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:

  1. Preferably loop region in known x-ray structure
  2. Use hydrophobicity plots to minimize hydrophobic residues at both termini
  3. 3-4 residue linker between protein and affinity tag (GST/6xHis)

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.

EDIT: I also played around with the PDB file of the non-truncated protein to check whether huge hydrophobic patches were exposed on deletion of the domain(s), and I didn't find any such patches that could potentially be exposed and lead to aggregation


1 Answer 1


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.

  • $\begingroup$ I used the x-ray structure to determine known loop regions that connect structured domain regions (pt #1) $\endgroup$
    – gkadam
    Commented May 10, 2012 at 12:53
  • $\begingroup$ I see - if this is a series of independent folding domains like SRC, what you have done makes sense. In this case what you call 'loops' would be more like linker peptides. In this case, sometimes things that look independent actually are not. Its also possible to get precipitated protein for more conventional reason - problems with heterologous expression or codon choice when moving from human to e coli for instance. its hard to be more specific without details. does seem as if you made a reasonable effort. $\endgroup$
    – shigeta
    Commented May 10, 2012 at 18:33

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