Hi,
I am trying to purify the chimeric fusion proteins containing NGF and various functional domains of Fibronectin. Although lower molecular weight proteins were secreted into the conditioned media (up to 70 KDa) and successfully purified, higher mwt proteins were not secreted (>75 KDa, 104 KDa and 132 KDa).
My question
1) What could be the potential reasons that not secreted
2) Which procedure can be useful to purify (6X-His-tag) these proteins
Thank you
Raju
Would you consider purifying the proteins from a cytoplasmic extract instead of from the medium?
Hi Adam,
Thank you for your reply. I would consider, however, I have purification issue with extracting from cells.
You could begin with metal affinity chromatography, thanks to the His tag, but this would probably not be sufficient to get the protein fully purified from Sf9 cells. You would then have to follow up with at least one more chromatography step. Affinity chromatography on a heparin-Sepharose column based on binding of fibronectin to heparan sulfate is one approach. Gel filtration chromatography is another, since the protein is quite large.
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