5 replies to this topic

[Givi]

I'm trying to express in E.coli and purify his-tagged protein. Problem, that worries me is relatively low level of solubility.
Currently I grow at +37, then make a "cold shock"(put flasks on ice for 10 min), induce with 0,15 mM IPTG (instead of 1 mM) and grow overnight at +18 C. What else I can do to increase yield of soluble protein?
I have two more questions:
2) while purifying protein I have noticed that I have more impurities (presumably DnaK E.coli chaperon) than my recombinant protein. Why it can happen?
3) How does culture "owerincubation"/"overgrowth" influence yield of soluble protein?
Looking forward to your reply

P.S. mistakes corrected.

Edited by Givi, 23 June 2009 - 11:08 AM.

[PhD]

Hi,

id never heard of heat shock increasing the solubility. Why do u do it for so long though? Is it a standard method in your lab?

I also am interested in replies to ur question since I have the same problem
[/quote]

[PhD]

Soory I meant cold shock, id heard of heat shock before but what does the cold shock do?

[Givi]

Does anybody know any SlyD deficient / knockout E.coli strains?
It seems that I have severe contamination both with SlyD and DnaK proteins from E.coli.

Edited by Givi, 24 June 2009 - 08:43 AM.

[swanny]

Givi, on Jun 24 2009, 02:41 AM, said:

I'm trying to express in E.coli and purify his-tagged protein. Problem, that worries me is relatively low level of solubility.
Currently I grow at +37, then make a "cold shock"(put flasks on ice for 10 min), induce with 0,15 mM IPTG (instead of 1 mM) and grow overnight at +18 C. What else I can do to increase yield of soluble protein?
I have two more questions:
2) while purifying protein I have noticed that I have more impurities (presumably DnaK E.coli chaperon) than my recombinant protein. Why it can happen?
3) How does culture "owerincubation"/"overgrowth" influence yield of soluble protein?
Looking forward to your reply

P.S. mistakes corrected.

You could try a different fusion partner, or an additional partner. There are lots of papers and reviews on ways to improve solubility of proteins. MBP is a favourite partner, as is NusA, but there are lots more. I'd suggest a short literature survey to see what other groups suggest. It might be worthwhile seeing what the structural genomics consortia are using; they want as many positives as possible from as early as possible.

[Givi]

Problem is that I cannot use any fusion partners as MBP or GST or others, because my protein forms oligomers and I cannot 1) digest completely fusion protein; 2) I cannot separate digested and  undigested fusion proteins using gel filtration chromatography. So I need to use really small tag like His-tag.
So I need other ideas for solubility improvements.