Protein purification after cleavage (thioredoxin) - (Jan/25/2007 )

hello
I work on a 24kDa protein
I purified this protein in fusion with thioredoxin-His-Tag without problem
I did a chemical cleavage and the SDS-Page gel show a good yield
My problem is that I can't separate my protein and thioredoxin
they have similar weight (near 20 kDa), similar charge and pI
please help me I tried affinity separartion (Ni2+-NTA) and HPLC and each time the protein is released with thioredoxin

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what was done with HPLC (cationic/anionic exchanger chromatograpgy, hydrophobe binding chormatography, gel filtration, chromatofocussing) and what was not tried? there are numerous separation possibilities beyond His-Tag-Ni2+ chelating, although I wonder why you co-isolate both AFTER cleavage; maybe they complex to each other which can be overcome by lowering salt or increase of detergent (triton x-100 f.i.) conc

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when I use HPLC or affinity chromatography both protein (mine and thioredoxin) are elued in the same time so I can't separe them
I would work with only my protein because I am not sure that there is not interaction between them
when I dialyse the proteins neither thioredoxin nor mine precipited they are soluble
but maybe I could try increasing detergent concentration why not
about gel filtration the proteins are not too closed ?
thank and if you have others ideas ...

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if using detergent in gel filtration conc should be below CMC which is also dependend on temp; co-elutions are an old problem in biochromatography and needs some trials; if you only need small amounts of your protein, may be enrichment by immunoprecipitation or (more preparative) immunoaffinity chromatography is useful;

IP at all may give you hints if really tag and protein have interactive binding; if so, some optimization of conditions for gel chromatography may be done with IP ...

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