Ion Exchange Column sugguestions!

[KAR10]

Hi All,

Ok Im working a fusion protein pI of 6.9 using a Phenomenex BioSep DEAE column 78x7.5mm. So Ive tried using 100mM Tris pH 8 and buffer B 100mM Tris + 1M NaCl pH 8; 100mM Tris pH 8 and 100mM Tris + 100mM NaCl pH 8; 20mM Tris pH 8 and 20mM Tris + 100mM NaCl pH 8 and the same last buffers at pH 8.5. This protein will not stick to the column for the life of me. Im diluting it in the mobile phase and running an isocratic separation for 10min at the start of each run before the gradient which is 100% A to 100% B in 50 minutes and it elutes at 5 minutes each time. Im running at a flow of 0.5mL/min. Now PD was having problems with the process and are using a Q Sepharose column for the Anion Exchange step in their process. Does anyone know of an analytical column similar to that? Any suggestions as to how I can get this protein to stick? Ive used this column for all my other proteins and never had a problem (they are all slightly different with much lower pIs ~5.5 or so. Part of the issue I suspect is that one end of the protein has a pI of approx. 5 and one has a pI of approximately 9. Please help! If you need more information please let me know:)

[Uwe Neue]

Your protein may have too weak a charge at pH 8, or even no charge. Since you tried already a weak starting buffer as well as pH 8.5, this may not work with a weak anion exchanger (since your weak ion-exchanger may loose charge at this pH already), and you are likely to need a strong ion-exchanger (Waters Biosuite Q AXC).

[Hassel Hoff]

The phenomenx biosep is a size exclusion column. If you protein is not being retained, it is too large to fit in any of the pores.

[LittleAlex]

Hi All,

i'd like to know if anyone has heard about an GE equivalent to the ProtEx DEAE Anion exchange (Supelco). I ve already try to contact GE, but im still waiting for their answer...

thanks for your reply,

Alexia )

[ALLEN HIRSH]

Hi All,

Ok Im working a fusion protein pI of 6.9 using a Phenomenex BioSep DEAE column 78x7.5mm. So Ive tried using 100mM Tris pH 8 and buffer B 100mM Tris + 1M NaCl pH 8; 100mM Tris pH 8 and 100mM Tris + 100mM NaCl pH 8; 20mM Tris pH 8 and 20mM Tris + 100mM NaCl pH 8 and the same last buffers at pH 8.5. This protein will not stick to the column for the life of me. Im diluting it in the mobile phase and running an isocratic separation for 10min at the start of each run before the gradient which is 100% A to 100% B in 50 minutes and it elutes at 5 minutes each time. Im running at a flow of 0.5mL/min. Now PD was having problems with the process and are using a Q Sepharose column for the Anion Exchange step in their process. Does anyone know of an analytical column similar to that? Any suggestions as to how I can get this protein to stick? Ive used this column for all my other proteins and never had a problem (they are all slightly different with much lower pIs ~5.5 or so. Part of the issue I suspect is that one end of the protein has a pI of approx. 5 and one has a pI of approximately 9. Please help! If you need more information please let me know:)

I hope you have solved your particular problem since last summer, but your post raises some interesting general points that are worth commenting on. As Dr. Neue correctly pointed out, it is likely that the weak anion exchange column that you worked with is strongly discharged, even at pH 8. Phenomenex, for instance, gives the effective pH range of the column as 2.5 to 7.5. Thus, switching to a strong anion exchanger and a higher binding pH, e.g. 9 -9.5, is a natural direction to go. Nevertheless, it seems that you don’t know enough about the physical properties of your protein, and finding its actual pI would be a good starting point. In your post you refer to one end of the protein having a pI of ~5 and the other end ~9. The concept of a double pI is meaningless because the actual measured electrophoretic pI is always the pH at which the whole protein molecule has an effective net charge of zero so it ceases to move in an electric field. Since your protein is a fusion protein, I assume that you actually referred to the measured pIs of the original two proteins as separate molecules. That, in turn, makes me wonder whether your estimate of a pI of 6.9 is your guess based an arithmetic average of the pIs of the two individual proteins. If so, it is not a reliable way to estimate the pI because one of the fused proteins may have significantly more charged groups in its sequence than the other and would thus dominate the overall charge total, skewing the fusion protein pI. Of course the standard theoretical method using the fusion protein sequence will usually yield an estimate within a pH unit of the protein’s pI and that is a good start. However, the protein does not bind to an ion exchanger with all its charges because it has a large rough surface with unevenly distributed charge. Instead, it tends, at any given pH, to rotate into positions with clusters of local surface charges oriented towards the opposite charges of the ion exchanger. This yields the most favorable binding free energy. This fact provides you with a very flexible tool for separating your protein. First bind it at an appropriate pH, usually 1 pH above the apparent pI on an anion exchanger or 1 pH unit below the apparent pI on a cation exchanger. Then you can elute the protein with a controlled pH gradient, using pISep (www.cryobiophysica.com). PISep is a new external pH gradient IEX chromatography which works smoothly on both cation and anion exchangers allowing the creation of ascending or descending pH gradients from pH 2.4 to 10.8 using a single buffer chemistry. By varying the starting pH, the pH gradient slope or adding isocratic salt you can selectively shift the apparent pI, the pH at which the protein elutes from an ion exchanger, over several pH units. The pI shifts are very sensitive to variations in both the protein’s isoform chemistry and its configuration, and chromatographic resolution is significantly increased when pH gradients are flattened, so you should be able to purify even very challenging proteins.