3 replies to this topic

[Goggi]

I would like to ask for the help in order to solve my problem.
I'm expressing human protein (a fragment of it, to be precise). When I'm analyzing expression/Ni-NTA purification by sds-page, I see 2 bands instead of one. I have performed trypsin digest/peptide fingerprinting by mass-spec. Both bands belong to my protein. The difference between bands is ~5kDa. I cannot separate degraded fractions by subsequent size-exclusion chromatography. I have checked my protein by PeptideCutter. There is a number of peptidazes that cut. However, I found that Asp-N endopeptidase could cut ~5kDa at carboxy terminus which corresponds to what I see on sds-page.
I'm really annoyed by this degradation problem and do not really know how I can solve it.
May be I should mutate the "putative" recognition site for endopeptidase?
The cleavage site which is given at Expasy is:
Xaa-|-Asp, Xaa-|-Glu and Xaa-|-cysteic acid bonds
The sequence of my protein is:
MRKEAEKTAL STIAVATAKA KEQETILRTR ETMATRQEQI QVTHGKVDVG KKAEAVATVV
AAVDQARVRE PREPGHLEES YAQQTTLEYG YKER
I would be extremely helpful is anyone could help me to solve this problem.

[mdfenko]

add protease inhibitors to the expression medium.

[Goggi]

Doesn't make sence to me, as pretease inhibitors will inhibit all bacterial proteases, thus, preventing cell from growing normally (I think cells will not grow/express at all). Does anybody else has another suggestion?

[mdfenko]

they shouldn't enter the cells but will inhibit the proteases that are released into the medium