Hi,
I would like ask your advice on designing SUMO fusion proteins.
Specifically, is it recommended to include a linker between the
C-terminus of SUMO (-QIGG) and the N-terminus of the target protein?
Figure 3 of Guerrero et al. (https://pubmed.ncbi.nlm.nih.gov/26297996/
<https://pubmed.ncbi.nlm.nih.gov/26297996/>) suggests that including a
linker of SGG or SGGG greatly improves cleavage efficiency by Ulp1.
However, the often-stated advantage of SUMO is that, in the absence of
a linker, the target protein contains no extra residues after cleavage.
As the author,
1) GGG is not required when the globular domain is not so close to the
Ulp1 cutting site. For example, if you have a flexible N-terminus, the
insertion like GG, GGG is irrelevant.
We used an Intein domain in this manuscript, the N-terminus is very
close to the core of the Hint fold. Without a short linker, Ulp1 does
not cut at all. The required length for cleavages
actually varies depending on different inteins.
if the cutting site is immediately adjacent to a well-folded region, it
will not be cut. I recommend 2-3 flexible sequence such as GG,GGGG. This
will change the efficiency of the cleavage.
you could also increase the temperature from 25 to 37 degree if one has
a problem with cleavage.
However, the fused proteins aggregates or form oligomers, preventing
Ulp1 to access, then again, Ulp1 will not cut and might need even a
longer linker for the cleavage.
I’ll also take any advice on your favorite N-terminal His tag for SUMO
fusions. I am considering MGSSHHHHHHG, based on two Acta F papers
(https://pubmed.ncbi.nlm.nih.gov/22949189/
<https://pubmed.ncbi.nlm.nih.gov/22949189/>,
https://pubmed.ncbi.nlm.nih.gov/18391421/
<https://pubmed.ncbi.nlm.nih.gov/18391421/>), or perhaps
MGSSHHHHHHSSG, which is from pET14b.
GSS tends to get the gluconoylation modification, which could be
annoying when you need exact Mass analysis as usually depending on
expression condition.
https://pubmed.ncbi.nlm.nih.gov/9918669/
My recommendation is to pay attentions to DNA sequences which could
affect transcription initiation or translation initiation. Commercial
vectors widely used are usually fine.
best regards,
Hideo
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