Ferritin purification by anion exchange

[yongyuandedj]

Hi all,
I try to purify ferritin from E.coli lysate and we found that the recovery is very low (10% only). So i use horse spleen ferritin (24 subunits) as a standard trying to find out the problem (I use Hi Trap Q column, 0.15M NaCl gradient). I found that there is one peak at around 0.1M NaCl. But when I regenerate the column using 0.5M NaOH, there is another peak coming out at the highest conductivity (about half mAU of the one during gradient elution). How come there are two peaks when i only load one type of protein??? In this case, shall I try to use higher gradient elution, say 0.5M or 1M NaCl to get higher recovery? Thank you~~~~

[leekaming]

Have you run SDS PAGE for the two peaks?

[smhall]

If you are following some kind of published protocol regarding how ferritin (I am guessing you have a recombinant in E coli host) elutes from strong anion exchange on a Q column, then I could understand why you only increase the ionic strength to 0.15 M. As a general rule, I would always make the B buffer to have 1.0 M NaCl and run a steeper gradient at the end, usually as a cleaning procedure. In fact, if you do repetitive runs, I would probably not even regenerate with 0.5 M NaOH if I really did not have to do so.

Poster leekaming suggests checking if the proteins have the same relative mobility, possibly indicating they are identical. However, one of the proteins might be missing a few residues on either the N- or C-terminal end, and SDS-PAGE does not resolve that. Your best approach is to treat the material from the two peaks, digest them in trypsin, and analyze on C18 HPLC to look for the profiles, taking note of peaks present or absent at the different retention times. Further protein chemistry techniques might account for difference in affinity to the SAX column, including estimating thiols and disulfides of Cys and cystine, and post translational modifications (phosphates?)

[yongyuandedj]

If you are following some kind of published protocol regarding how ferritin (I am guessing you have a recombinant in E coli host) elutes from strong anion exchange on a Q column, then I could understand why you only increase the ionic strength to 0.15 M. As a general rule, I would always make the B buffer to have 1.0 M NaCl and run a steeper gradient at the end, usually as a cleaning procedure. In fact, if you do repetitive runs, I would probably not even regenerate with 0.5 M NaOH if I really did not have to do so.

Poster leekaming suggests checking if the proteins have the same relative mobility, possibly indicating they are identical. However, one of the proteins might be missing a few residues on either the N- or C-terminal end, and SDS-PAGE does not resolve that. Your best approach is to treat the material from the two peaks, digest them in trypsin, and analyze on C18 HPLC to look for the profiles, taking note of peaks present or absent at the different retention times. Further protein chemistry techniques might account for difference in affinity to the SAX column, including estimating thiols and disulfides of Cys and cystine, and post translational modifications (phosphates?)

Hi thanks very much for your suggestion. i will try them. Besides, actually the published protocol use weak anion exchange (DEAE)for purification but i don't have that one in our lab. i wonder whether it could be due to the lower selectivity of strong anion column?

[leekaming]

Poster leekaming suggests checking if the proteins have the same relative mobility, possibly indicating they are identical.

I suggest to run SDS PAGE for the two peaks is to see whether both peaks contain your protein. One protein in and two peaks out do not mean that your protein separate into two peaks.
Very often the NaOH wash result in peak but contain impurity other than your desired protein.