I have cloned a beta-lactamase gene in BL21 E.coli strain using pET28a vector for expression and in vitro studies. After Ni-NTA column purification of this His Tagged protein, I am getting two bands in SDS-PAGE gel instead of one. What could be possible reasons? I have checked the activity of the protein and it's working absolutely fine.
Hi Gaurav,
I had the same case as yours 4 years ago. I cloned a gene and overexpressed the protein in E.coli. Two bands with a similar density appeared in the SDS-PAGE after induction by IPTG. Both bands existed despite of further purification. I thought it might be protein lysis so I sliced both bands from the gel and tested by mass spectrometry. However, both bands corresponded to the intact protein!
Finally, I found something tricky in the SDS-PAGE loading buffer resulting in the 2 bands. I used 2-mercaptoethanol as the reducing agent in the loading buffer at the beginning, but when I used DTT instead of 2-mercaptoethanol, the extra band disappeared.
I don't know if this is the key in your case, you may try using a different reducing agent. As your protein activity is fine, I believe your case is just also from a trick in SDS-PAGE.
Good luck!
It would be better if you can provide following details
Cloning sites and size of the bands obtained etc.,
Anyway first analyze the beta-lactamase gene for presence of any internal ORFs and/or proteolytic sequences.
As pET28a vector contains HIS tag at both ends; it may not separate truncated products.
If you find any internal ORFs in the gene; then you can avoid truncated products by using the 5' HIS tag purification (By introducing STOP codon before 3' HIS tag/ changing the vector)
In the case of proteolytic sites; the recognition site needs to be silent mutated or use protease inhibitor cocktails.
Is the 2nd band larger or smaller than the expected molecular weight? If smaller, it could be that your protein was clipped by protease. If larger, it could be caused by reading through the stop codon. The first problem might be fixed by increasing your efforts to avoid proteolysis by (a) switching to a different strain of E. coli, (b) including protease inhibitor cocktail during extraction and purification, (c) keeping everything cold during purification, and (d) working as quickly as possible. The second problem, can be fixed by adding tandem stop codons to the vector.
Another explanation is that there is a contaminating protein in your prep. You may need to run a second chromatography step to get rid of it.
you are cloning beta lactamase, an enzyme. Isoenzymes will migrate with similar velocity and will be slightly different in size.
http://europepmc.org/abstract/MED/12879
I hope it helps. Have a wonderful day
Your protein could be dimerizing. Of course SDS implies denaturing conditions, but certain proteins such as sigma factors do show multiple bands on SDS PAGE and Westerns.
Hi Gaurav,
I had the same case as yours 4 years ago. I cloned a gene and overexpressed the protein in E.coli. Two bands with a similar density appeared in the SDS-PAGE after induction by IPTG. Both bands existed despite of further purification. I thought it might be protein lysis so I sliced both bands from the gel and tested by mass spectrometry. However, both bands corresponded to the intact protein!
Finally, I found something tricky in the SDS-PAGE loading buffer resulting in the 2 bands. I used 2-mercaptoethanol as the reducing agent in the loading buffer at the beginning, but when I used DTT instead of 2-mercaptoethanol, the extra band disappeared.
I don't know if this is the key in your case, you may try using a different reducing agent. As your protein activity is fine, I believe your case is just also from a trick in SDS-PAGE.
Good luck!
Thank you all for your response.
@Suneelshekar- I used NheI and SacI sites. I did not put stop codon in the reverse primer.
@Adam- The second band is slightly higher (approx 1-2 kD) than desired one. Your explanation has the probability get two bands but I am amazed.
@Maria del- I am working with a purified protein. How can I get isozyme in my sample? I can not access to full paper, can you please provide me?
There are possibilities of two bands just because your protein may be dimer, you can go for the native PAGE and compare it with the SDS PAGE. The picture may clear whether the protein get denatured or not.
Some of the protein might be cleaved. Check the sequence if there is any protease site.
Everyone provided very helpful suggestions. But your solution will all depend on the size of the bands. In your question the most important fact is missing, is the extra band larger or smaller than your expected size. This will narrow down to which trouble shooting method suggested above to start applying to get rid of the unwanted band.
@Eun D. Lee- I am getting extra protein band higher than expected size(approx 2kD) along with protein of my interest. I am going to do purification again.
Earlier I set up expression check at lower volume (10mL) to confirm accuracy of my experiment. After that, I went for scale up to 1 litre volume and got two bands instead of one at 200mM imidazole elution.
Hi Gaurav,
According to your further description, I believe that you have run into the same problem as mine. I attach a picture of my SDS-PAGE gel here, in which two bands (indicated as red arrows) were generated after IPTG induction. These two bands appeared only a few kD difference. Same as your case, the lower band corresponded to the real MW of my protein, and the upper band is the extra one.
I solved this problem by using DTT instead of 2-Me in the loading buffer as I described in my previous answer. A student in my previous institute also ran into this situation when he was working on protein purification and he also solved this problem after I told him to try using DTT in SDS-PAGE loading buffer. Thus I strongly recommend you to try running SDS-PAGE again using another loading buffer before you repeat your purification.
Hi there,
Is there a signal peptide encoding sequence in the cloned gene? If yes it might explain the presence of mature and proenzyme forms... Where is the tag? Pass the lysate on NiNTA to know where the cutting occurs.
The higher protein could be your protein plus signal peptid if you have signal sequence.
Hi Elham and Dominique. I have removed the signal peptide from N-terminal region and His tag is present at N-terminal region.
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