Dear Sara,

I suggest you to check if your protein is in the soluble fraction after bacterial lysis. This will be important because you can improve the solubility by changing the growth temperatura ou buffer pH (which depends on pI of your protein). If you are purifying by affinity and then size exclusion column you can check the protocols in this publication doi.org/10.1105/tpc.20.00297 and manual https://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Sigma-Aldrich/General_Information/1/ge-strategies-for-protein-purification.pdf. You can test these conditions by purifying a small scale of your protein induction.

In case you are doing ion exchange purification, it will be a different protocol with A buffer without salt and elution B buffer with salt. Also, the buffer pH will depend on your protein pI. Good lucky.

Hi Sara,

Can you tell a bit more about the proteindd you are going to purify? How pure do you need them to be and in what scale are you working?

Usually a 3-step combination of different chromatography resins/techniques will work. Tell me more and I can give more detsils.

Best regards, Anna

Cytiva Life Sciences offer some very good guides to protein purification (https://www.cytivalifesciences.com/en/gb/solutions/protein-research/purify). These include some overviews and then details on specific techniques.

As Anna Heijbel suggested, if you could give some more details then we can suggest specific tips.

Good luck!

In order to give you helpful advise we need more information. Proteins are very different! Are you looking at purifying a native protein? What is the source? Or is it a recombinant protein? Perhaps with a tag? The approach will be different depending on the source. And as Anna mentioned above your purity need and scale is also important to consider. Assuming a his-tagged protein expressed in E. coli in soluble form Ni-IMAC followed by anion exchange would be my first choice. Perhaps followed by size exclusion.

And do let us know where in the process it goes wrong. Do you loose a lot of material in some step? Or is it not pure enough? Do you see a lot of aggregates? Etc.

It is a good idea to check the known properties of the protein or similar proteins in the literature and if te sequence is known look at properties such as isoelectric point. That can give you clues to which conditions your protein will accept and which techniques to choose.

Processes for purifying proteins are almost custom-made for every single protein. We really need a lot of more information to be more helpful. Expression system? What are you purifying from, and to? State of expression? Protein size, shape, and pI, post modification state? Tags? Native or denatured?

Hello everybody,

Thank you for all your helpful comments.

Here is the necessary information:

Protein A: 24 KDa, pI; 5.33

Protein B: 13 KDa, pI; 6.57

Both of these proteins have been expressed successfully in BL21 and they are soluble.

I extract them under native conditions and they also have His-tag at N-terminal.

However, I have difficulties in purification.

I always run on a gel an aliquot of the sample before passing through the nickel resin, as well as an aliquot of the flow-through and washing steps.

I elute the protein of interest by elution buffer and using imidazole step gradient.

But unfortunately, I did not see any elution peaks and it seems that protein of interest does not bind to resin and they always exist in flow-through.

I guess I should change something during injection or modify some elements in buffers. So, I look for a good buffer recipe and purification protocol using AKTA.

I have attached a PowerPoint containing the photos of SDS-PAGE.

All suggestions are welcome and appreciated.

Sara, looks like too many unspecific contaminants are binding to the IMAC column. You could try an ammonium sulphate (AS) pre-fractionation before going on the ÄKTA, add solid AS to about 20, 40, 60 and 80% saturation and see where you get. Also make sure, your NTA column is stripped and fully charged with nickel before each run, to ensure defined and optimal conditions.

Hi Sara,

Have you tried other metals for your purification of your His-tagged protein A?

Usually a cobolt charged resin will give you higher purity (less contaminants are binding).

I think you should test other metal charged resins for your purification. This is often forgotten, but can be a great help. There is a kit available for this purpose with 4 x 1 ml prepacked columns where the resins are precharged with Ni, Co, Zn and Cu. You can find the kit here, www.bio-works.com/product/imac-resin/babybio-his-tag-screening-kits

We usually work with Binding buffer: 50 mM sodium phosphate, 300 mM NaCl, 10 mM imidazole, pH 8.0

For elution we start with a linear gradient 10-15 column volumes with Elution buffer: 50 mM sodium phosphate, 300 mM NaCl, 300 mM imidazole, pH 8.0

When we see in the gradient how high imidazole concentration we can include in the Elution buffer and still have the His-tagged protein bound we use this higher concentration in our Binding buffer for the next purification of the same protein and it will get higher purity.

Now you have optimized on metal ion used and concentration of imidazole!

Remember to you an imidazole of high quality. The salt should be white and not torwards yellow.

Please, contact me if you need more help, [email protected]

Hello again Anna Heijbel, Boopathi Subramani

Thank you for all your helpful comments.

I never tried other metals and I always use HisTrap™ HP from GE healthcare. I resuspend the cell pellet (from 600 ml of culture) in 5 ml of lysis buffer. Then I add lysozyme and sonication, etc. Afterward, I try purification by AKTA. I attached the file including the recipe of all buffers and steps of protein purification. I would really appreciate it if you could let me know whether there is something wrong with the composition of buffers or the steps of purification.

Kind regards,

Sara

Hi Sara,

From the protocol and elforesis gels you sent earlier it looks like you have most of your target protein in the flow through, A3 fraction. Is it correct?

I also see that you have b-MEA in you buffers. Having this reducing agent in the buffers may get it harder to bind your target protein as it usually drag out the nickel ions from the resin. Have you seen any change of color of your resin (getting lighter blue?. If you see this you should use a resin that is developed to be resistant when having reducing and/or chelting agents in your buffers. Then I suggest you instead use the BabyBio NiMAC columns (available in 1 ml and 5 ml sizes) that are prepacked with a specific resin resistant to reducing and chelating agents (up to 20 mM).

If you don't think, what I describe above is a problem, my suggestion is to test different metal ions, as I said before this can easily be done by using the BabyBio NTA His-tag Screening kit.

Binding buffer: 50 mM sodium phosphate, 300 mM NaCl, 10 mM imidazole, pH 8.0

Elution buffer: 50 mM sodium phosphate, 300 mM NaCl, 300 mM imidazole, pH 8.0

Linear gradient, 15 column volumes.

From the result in the linear gradient, when you see your target protein eluting you see the concentration of imidazole you can have in your binding buffer and still have your protein bound. For highest purity it is always best to have as high as possible concentration of imidazole in the binding buffer to prevent impurities from the cell to bind.

If you instead know that your target protein is in the flow through, you should remove the imidazole from the binding buffer (or decrease the concentration).

Maybe you also have to add a second step, an ion exchanger and/or size exclusion.

I'd agree with Anna Heijbel suggestions for amending your protocol. They are all sensible.

In particular, raising the pH will help a lot. The pI for histidine protonation is just below pH 7, which means that at pH 7.4 a proportion of histidines will be protonated. Protonated histidines will strongly repel from nickel rather than chelate to it. Phosphate buffer pH is very stable with respect to temperature, so you are probably working at too low a pH. Raising to pH 8.0 should help - that should mean that histidines aren't protonated.

Unfortunately it is sometimes the case that His-tags fold back into the protein and are masked from nickel. As you have very strong soluble expression - your protein of interest is one of the two most abundant proteins for protein A at least - you should also consider ion exchange as a primary purification step. This will need a little more optimisation, but it is very likely to be successful in your case.

I'd raise another point. Ekta is a difficult beast to work with and maintain, and in the case of histrap which has rather weak support, if the lines are not clean and you have pressure buildup you could easily bust the column. If you have some loose nickle resin lying around, I'd do a preliminary in vitro check that the protein actually bind. Just chuck 20ul of beads in a tube with 200ul of protein, spin, wash with whatever you wash, and straight to PAGE by boiling the resin in 2X SDS loading buffer. In my experiences pH 7.4 is probably enough, but each protein is a case of its own and it may very well be the protonation problem Nicholas mentioned. In any case the above method is an easy way to check that your protien binds and you're not wasting machine run time and pump life.

Good thing is that histraps are cheap. The small one is even syringe adaptable, so you don't even need the akta if that's the issue. A handpressed hitrap run will be enough workout for the week though.

Hi,

Just to tell you that the newer (similar to HiTrap columns) prepacked BabyBio 1 ml and 5 ml column from Bio-Works that are available with many different IMAC, IEX, Protein A and more resins are even cheaper than HiTrap columns. They also often have higher binding capacity.

Hi Anna Heijbel Ian Hu Nicholas J Harmer and other friends,

Thank you for all your helpful comments. I modified the composition of buffers and some steps and the problem of protein purification has been solved finally!

Here are some changes:

1. Removing imidazole in lysis buffer.

2. Removing β-mercaptoethanol from all buffers (lysis buffer, wash buffer, and elution buffer) and increasing the pH of all buffers to 8 (I prepared buffers according to QIAGEN booklet).

3. Resuspending cell pellet (from 600 ml of culture) in 20 ml of lysis buffer (instead of 5 ml).

4. Washing new columns with MiliQ water and then equilibration of the column with lysis buffer and applying the sample afterward.

5. Reducing the flow rate from 1 ml/min to 0.4 ml/min.

Hi Sara Heidarpanah ,

Congratulations on solving your problem - I'm really pleased that you've been able to get your purification sorted out.

Thank you also for sharing your solution to the problem. I'm sure that this will be helpful for others.

Good luck in your research!