I have cloned a mammalian protein in pGEX vector (GST tag) and transformed it in Rosetta 2 cells. I could get good expression earlier and used 1 ml of beads (washed and suspended in PBST) however the recovery was very low (I could only get 30% of the expressed protein bound to the beads). If I increase the beads, I am getting non specific bands. Could anyone suggest a way to to increase the yield?
Another problem is, now I can not get expression as I did previously. What could be the reason. I have used cells from glycerol stock also for expression.
The conditions of expression:
IPTG: 0.3 mM
Temperature: 15 for expression
Time: about 12-18 h for expression
Please suggest some solutions for these two problems.
In terms of the proteins I work with I have seen "soluble expression" and poor binding to the affinity matrix and in the end I found that the protein wasn’t correctly folded and thus didn’t bind to the beads.
In terms of the expression I have found a fresh transformation each time works really well (I have also seen glycerols dropping expression over time), I then dissolve up the plate (all of cells) with media and then use this as my inoculum. I then tend to start with a non IPTG induction protocol, this is easy and you don’t need to fuss about induction OD I tend to run the expression at 30oC for 24-36h but you can reduce the temp further if required
http://www.bioc.aecom.yu.edu/vetting/PROTOCOLS/noniptg.html
James
Hello,
I agree with Paul View.
The problem could be with your glycerol stock, if you are using the same condition as before.
Or, The problem could also be with your beads. You can wash your beads (Ni-NTA) (in case you are reusing your beads) with 2M imidazole and then you can charge beads again with 0.1M NiCl3. this helps you to get better yield.
Third, Non specific bands. If your protein expression is quiet less, then most likely bacterial highly expressed chaeperone proteins (E.coli PPI) will compete with your protein to bind with Ni-NTA and you will end up with higher fraction of your protein in unbound fraction. If this is the case you can try with Cobalt based talon resin, where E.coli PPI protein doesnot bind and you will get rid of non.specific bands with better yield of your protein.
Use freshly prepared antibiotic. If you use old chloramphenicol then the expression level can decrease.
Hi All,
Thanks for the suggestion. I have made fresh transformation in Rosetta 2 cells and now getting same production. Regarding the yield, I am getting the protein in second elution (earlier did only one elution) and is higher than the first elution. I hope this yield is enough for my experiments.
Thanks for James and Paul
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