Editing Protein Purification 3 Steps (section)

=== Capture  ===

<br>Affinity chromatography (AC), Ni<br>Ion exchange chromatography (IEX), SP FF. 

<br>Centrifugation at 45,000 rpm for 30-45 minutes is sufficient for most proteins (you can live also with half of it using SS-34 rotor at 20,000 rpm for 2 hours). 

If using a dripping column, initially pack beads that will be sufficient to ~10mg protein/1L of induction. After the first purification it will be easier for you to adjust the amount of beads to get the best purification without getting too much junk. 

Remember that in most cases the affinity chromatography step will determine the fate of your sample in terms of purity levels. Do your best to calibrate this step to the optimal setting possible so you get the protein at the highest purity level. From my own experience a dirty sample post affinity will not improve tremendously after using other columns (again, in most cases). 

Perform extensive washes, especially if working with dripping columns, 20 times the amount of beads volume (20 CV), varying the amount of salts and Imidazole. 

Elution should be conducted at a relative small flow rate to get the sample concentrated at the end of this step; in certain cases you’d want to increase the flow rate to avoid high concentration elution in case the protein is prone for aggregation. 

Evaluate your sample purity through an SDS-PAGE analysis. In case you find it hard to identify your protein do a western blot for verification (blot directly from the coomassie-stained gel). 

Depending on the purity level required and the observed purity of the sample, assess whether you want to do a step of ion-exchange or size exclusion. 

In case you plan on doing a step of ion-exchange, you might consider eluting your protein with low salt concentration (maximum 50 mM ion strength) and continue directly with your post-affinity elution into the ion-exchange column. 

<br> 

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. 

<br> 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. 

*Spin down cells, then suspend cell pellet with 100 mM Tris-HCl or Tris-Acetate (pH 8.0) containing 20% sucrose and 0.5 mM EDTA and incubated on ice for 15 min. 
*Spin down cells again, the supernatant (sucrose wash) contains periplasm proteins.
*Resuspend cell pellet again in ice-cold deionized water and incubated on ice for 15 min. 
*Spin 6,000 rpm for 5 min, the supernatant is combined with supernatant from the sucrose wash---the combined fraction has most of periplasm proteins.

<br> Guanidinium/Urea wash – This is a bit risky but it is worth the try if protein is for structural studies. 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). <br><br>