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Unable to express protein in full-length form
Asrar Alam
Wednesday, 14 May 2008 11:01 UTC
I am expressing an enzyme in E. coli BL21
(DE3) which I am unable to express in full-length form. I am suspecting it is cleaved and degraded by some E. coli protease or it is getting activated and cleaving itself. Can anybody suggest a method by which I can get that protein in full-length form?
Thanks and regards,
Asrar Alam
Updated 14 May 2008 11:59 UTC
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Replies
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Hello
Did you try to express this protein in mamalian cells…293T cells or jurkat?
Regards
Raphael Roduit -
Thanks,
I haven’t tried expression in mammalian system but I am trying it in baculovirus expression system. -
hi
have you checked for activity? because once i was purifying my protein, the stop codon wasnt strong enough so I had many lengths of proteins and did think that my protein was being cleaved. -
Have you also tried including protease inhibitors in your solutions? If you do not express many proteins you should probably read this paper from structural genomics: PMID: 18235434
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As they said before, add protease inhibitors—PMSF/AEBSF or Complete tablets, benzamidine or even a mixture of them. Add them when homogenizing your cells, add again after breaking your cells, and add them again if you’re dialyzing at the end. You should also try doing your purification on ice or in a cold room.
If you have a tag on the C- or N- terminus, you can do a western to determine which end is degraded.
To test if it’s degrading itself… knock out the active site residue(s) of your protein and see if you still have this problem.
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