Dear Imre, dear Katya,
thanks a lot for the prompt and insightful replies.
@Imre: well, I don't think it is a matter of linker of lack of Met/presence of stop codon after GST.
We use the exact same linker in bacteria and that works just nice, while wrt to the Met, we have cases in which we have kept the original Met (gene starting from aa 1) or in which we didn't include it (N-term deletion mutants) and we experience the same problem.
I tend to second rather Katya's opinion that what we purify in massive amounts are indeed native GSH-binding proteins (I dind't think at that at all!).
@Katya: I will have a look at the paper you sent me. However, the trouble is that the high amount of GHS binding protein is lowering the amount of GST-fusion protein we manage to recover from beads. So even being able to purify it form native GSH-binding proteins will not be ideal.
I think I will carefully consider the option of expressing MBP-fusion proteins, and move away from GST, as you both suggest.
Thanks a zillion for the tips,
best,
Sebastiano
On Mar 16, 2012, at 2:40 PM, Imre Berger wrote:
Dear Sebastiano -
I have actually no experience at all with GST in insect cells since I do not use this tag (mainly because I do not like the dimerization propensity of this tag and the glutathione elution step).
However, I do not think what you observe should be related to insect cells...but I have no data on my own for GST fusions in insect cells.
May I ask: Do you have a starting methionine in your construction AFTER the GST (i.e. did you keep for example the native ATG start codon when you cloned your gene?). And: Did you verify the reading frame (basically an huge excess of GST would mean that there is no readthrough resp. a stop codon somewhere close to the fusion tag, that is very unlikely.
Is there a long unstructured linker between GST and your protein resp. doyou suspect there is a long unstructured region in the N-term of your proteins?
I am soprry that I can';t help you much, but as said, Idon;t like GST - I used it as an undergaduate many years ago and totally disliked it (i had a protein that dimerized and with the GST tag which also dimerized it became an agregating mess), kept this aversion until now.
How about MBP? That's EXCELLENT.
On Mar 16, 2012, at 2:13 PM, Katya Heldwein wrote:
Dear Sebastiano,
what you see is not your cleaved GST tag but rather native insect glutathione-binding proteins that are produced in relatively large amounts (a few mgs per L). I am attaching a paper where recombinant GST-tagged proteins were successfully separated from these pesky insect GlutBPs under specific elution conditions. This has not worked for us, though. So, we simply do not express GST-tagged proteins in insect cells.
Good luck,
Katya
Ekaterina Heldwein, Ph.D.
Department of Molecular Biology and Microbiology
Tufts University School of Medicine
136 Harrison Ave
Boston, MA 02111
Sebastiano Pasqualato wrote:
Dear CCP4ers, Dr. Berger,
we have an accumulating series of GST-fusion proteins here that are all displaying the same behavior when expressed in Hi5 insect cells with the MultiBac system.
What we are experiencing is a massive production of free GST and only a limited amount of fusion protein.
Since the linker we have engineered between GST and our protein is the one that exists in the pGEX-6p vector series, with the preScission protease site, I was wondering if any of you had experience of this cleavage site being processed within these insect cells.
Thanks in advance for the help,
ciao,
Sebastiano
--
Sebastiano Pasqualato, PhD
Crystallography Unit
Department of Experimental Oncology
European Institute of Oncology
via Adamello, 1620139 - Milano
Italy
tel +39 02 9437 5167
fax +39 02 9437 5990
