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Purify, purify and…purify more: tips for improving your protein purification capabilities – part 2

In the previous post I have focused mainly on preparing and performing the first steps in the purification of a recombinant protein. In this section I will discuss a bit further about Nickel affinity chromatography via prepack column, about gradient elution and give some advice on improving purification of dirty preps.

Nickel affinity column gradient elution – capturing and purifying

While batch binding and elution step can be performed on a faster time scale (hour to hour and half from start to finish including wash steps), using a prepack nickel affinity column (such as HisTrap from GE) can help ending your first chromatography with a higher level of purity. This is true for all samples but it is crucial when you find your nickel affinity purification relatively dirty.

Why not use the prepack column from the first place?

It depends on the amount of proteins your purifying at a single moment (in our lab we sometime purify two to four proteins in parallel) and of course, if your sample volume is large it will take more time to bind, wash and elute.

When I am using a prepack column with gradient I still get the protein dirty no matter how much wash steps I do – what can be done to improve the nickel affinity purification?

From my own experience, when you have a good overexpression (i.e. higher than 15mg/1L of induction) then you will have better purification because the dominant protein in the sample will be your protein. However, if you have a low expression from the first place then your protein will have to compete with other host proteins which have similar expression levels. In such cases you should first consider improving expression via Autoinduction media (Studier et al. 2005) which I found to increase the fold of protein expression by 3-10 fold, depending on the protein. See my previous note about increasing expression.

Assuming you’ve tried everything and your protein is still low expressing you can do the following combination strategies, separately or combined:

  • Decrease beads volume – if using a prepack column, try loading the protein on a 1ml column and not a 5ml. Why do that? Well, let’s assume your protein is expressing at 5mg/1L of media and you induced 4L of culture, giving a total of 20mg of protein of interest. If you will load the lysate on a 1ml of prepack column, that can capture 20-30mg of tagged protein, between 67% to 90% of the column will be captured by your protein. On the other hand, if you load the 5ml prepack column, with a potential capacity of 200mg (!), all the rest of the host proteins will have plenty of room to bind to.
  •  Removal of periplasm – Another strategy is to do periplasm wash of the bacterial pellet before performing the bacterial cell lysis. That’s because the periplasm was shown to contain many proteins and enzymes that naturally bind divalent/metal ion. By removing these protein you lower the host protein’s that compete over binding to the beads.
  • Guanidinium/Urea wash – This is a bit risky but it is worth the try. With this strategy cells are lysed and bound to the nickel column. Then a wash with 2M of Guanidinium or Urea is performed which usually removes most of the host proteins and afterwards a shallow gradient of decreasing amounts of Guanidinium/Urea brings back your protein to native state. Of course, you will need to check and see that your protein didn’t loss its fold and function (usually if the protein refolds improperly you will notice aggregates once you concentrate your protein). The advantage of this technique is the ability to get a highly pure protein within one chromatography step.

Next I will discuss Ion exchange and Size exclusion chromatography

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