Recombinant nucleocapsid protein expressed in E. coli with N terminal His10 tag (expected size 45 kDa ).
Protein purified on IMAC crude FF washed with 250 mM imidazole/500 mM NaCl (pH 7.4) – not a clean profile on SDS PAGE
Protein applied to SEC S200 (150 mM NaC,l pH 7.4)
SDS Page & WB analysis of SEC indicates 3 major peaks
1. The first peak at >670 kDa (high order aggregate) – high level of DNA A260/A280 ratio faint Coomassie stainable band.
2. The second and major peak at slightly greater > 158 kDa on SEC (oligomer of N) contains two coomassie stainable bands on SDS-Page at 45 kDa (α-His positive) and 60 kDa band (non α-His positive) – co purified contaminant?
3. Minor peak containing 2 bands (approx. 20 kDa – non α-His positive).
I need to separate the His10 tagged 45 kDa protein species of interest (>158 kDa oligomer), which also probably has tightly associated E. coli DNA and RNA, from 60 kDa species.
To further clean up – I thought that trying IEX chromatography might be worthwhile.
The pI of monomeric 45 kDa protein species of interest is 7.1 however as it is likely to have DNA tightly associated I assumed a more negative charge and assumed Anion exchange MonoQ might be successful.
Wrong – desalted protein into 20 mM Tris pH 8.5 50 mM NaCl and applied to column with gradient (0-50 % B 20 CV, 50-100 % B 10 CV B contains 2 M NaCl),
· Maybe the protein precipitated when I desalted into 50 mM NaCl – Maybe I could start at 100 mM?
· Maybe I could repeat the IMAC on the IMAC/SEC purified material (i.e. 2 IMAC steps in total)
· Use a smaller IMAC column ( Increase the imidazole in the wash buffer)
· Sequence the two coomassie stainable bands and see what is potentially co-purifying & design a new purification strategy.
Any other suggestions would be greatly appreciated.
If your protein requires high salt to remain soluble, you could try hydrophobic interaction chromatography, which starts at high salt and elutes by a decreasing salt gradient.
For SEC line item #2: SDS-PAGE run reduced versus non-reduced? Answer may heip in diagnosis
For IEX: Was MonoQ column cleaned with caustic and/or guanidine showing material balance on gels analysis? Try slighltly acidic pH in flow-through mode? Use DEAE bonded pase? Answer may help diagnosis
For >158kD peak: Benzonase or RNAse sensitive if nucleoproteins not forming normal nucleocapsid? Any post-translational modification of wild type nucleoprotein from native host? Any cysteines in nucleoprotein sequence? 60kD may be heat shock protein removable by ATP wash of IMAC, or more MgCl2 in growth medium, or addition of denatured E. coli proteins to lysate?
It's a bit unorthodox, but if that nucleocapsid protein has no disulfide bridges and is relatively well behaved, I would try purifying it by denaturing IMAC (in the presence of 6 M GuHCl, for instance) and then renaturing it later. Its interaction with nucleic acids most likely depends on basic surface patches that will disappear upon denaturation (thus eliminating those aggregates). Most host contaminants interact with IMAC columns through surface His patches that also disappear when denatured, so those should be eliminated as well.
You could also try to add DNAse to your lysis buffer or, even cheaper, some basic substance (poly lysine, spermidine or some other polyamine). The idea is to get your nucleic acid either to be degraded by DNAse or to make it stick to the polyamines instead of your protein. Both strategies worked for me when I was purifying an extremely basic recombinant protein from yeast..
Good luck
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