I am trying to purify a protein dual tagged (HA and 6XHIS at c-terminus) from E.coli (BL21 DE3) using Ni-Nta column. The protein yields are low even when purified using 2L culture volume and also on concentrating, the protein precipitates interfering with activity assay. Can someone suggest ways to increase yield and also prevent precipitation? I have tried different IPTG conc. to check for increase in induction but no effect was observed.
You have several problems here, not necessarily unrelated. One is low expression levels (as suggested by the fact that you have been trying to optimize IPTG concentration). If you want help with that, more context is needed. Problems there may be caused by many things, ranging from poor practices during culture to suboptimal construct design.
Another is perhaps poor yield during purification (you do not provide much data here, either). Check aliquots of your entry sample, unbound fraction, eluate, etc. The objective is to find out whether the protein is binding to the column in the first place, and if it is, whether it is being eluted or not under the right conditions. A trial run under denaturing conditions -even if it doesn't meet your purposes down the road- can help discerning whether problems are stemming from in-column aggregation, poor exposure of the tag, etc.
Yet another problem is the precipitation during concentration. Solutions there are protein-specific. Try to keep pH as far as practically feasible from the pI of the protein. Play with different glycerol concentrations, or mild detergents. Try using L-arginine as additive, if at all possible. There's plenty of data in the literature about conditions you might try to explore, but this part is labor intensive any way you look at it.
Hope it helps!
Thank you for the valuable suggestions Alejandro Martin.
Yes in terms of expression level, not much significant difference was observed in induced vs uninduced sample tried over different conc. of IPTG and different temp. conditions. The construct is however correct checked by sequencing, cloned in pET21 vector also western blots showed protein at correct size.
I am able to elute my protein at 100mM Imidazole along with non specific high MW proteins but the yield is low. I also checked the Flow through and washes (20mM, 40mM imidazole) not significant protein band (corresponding to my protein size) was observed in any.
I have not tried with denaturing conditions yet as I needed protein in native conformation but yes that's a good suggestion to check for tag accessibility and aggregation. I will try to check if binding can be improved by adding denaturant.
Thank you.
Hi there,
If the protein precipitates during the concentration process, your buffer's composition is not ideal for your protein. The 3 main parameters to play with : pH, nature of the buffering agent and ionic strength.
Purification of Dual tagged protein from E. coli using Ni-Nta column :
1) You should optimize your Imidazole concentration to eluate protein from the Ni-Nta gel. May be you should use 200, 300 or 500mM of imidazole
However, you have to test the bioactivity in your brut extract (centrifuged culture) and compare it to the purified protein solution. Did you have a loss of activity after the purification column?
2) In our experience we purified His/tagged protein on Ni-Nta gel in a bach procedure: 1 hour incubation of the concentrated cloned protein culture. The supernatant was concentrated by ultra-filtration with a Biomax 10 kDa cut-off Centriprep device (Millipore Corporation, USA. The concentrated protein solution was incubated with the Ni-Nta gel for 1 hour with a low stirring and therefore the gel was centrifuged and washed with 1 volume of metal chelate affinity chromatography (MCAC) buffer 40mM imidazole. Protein was than eluted with Imidazole gradient steps: one step at 100 mM Imidazole and a second step at 200 mM and a last one at 300 mM.
3) You may also purify your concentrated protein extract on a Ni-Nta column but you should use a low Flow rate 5 or 10 ml/hour using the same gradient step to protein elution
4) Could you explain what is the objective of using a dual tag (HA and 6XHIS in the same c-terminus region?
The HA tag YPYDVPDYA epitope contains two Prolyl residues. We know that prolyl residue may lead to a repliement in the epitope and create a rigid structure that could mask or cover the His tag and limit your purification on Ni-Nta column
hi Mohamed, Thank you, I have not tested activity in crude extract but only with purified protein.
1) I have tried increasing Imidazole conc. upto 300mM but more non specific bands are observed when increased beyond 100mM.
4) The purpose of using dual tag is to eliminate false positive as bacterial proteins also have hisitidine hence on detection of my protein which is heterologous it may interfere and give the false positive signal. His tag is used for purification while HA tag for specific detection.
- Badges
- Science topic
- Similar topics
- Microbiology
- Assays