I'm working on Novel protein therapeutics for peripheral nerve regeneration. My protein NGF (Nerve Growth Factor) was expressed in Sf9 cells and secretory protein was unable to purify under native or denature conditions. I have performed Ni-NTA agarose resin and followed by silver stain and western blot. Other side I also tried to purify using Q-Sepharose ion exchange chromatography followed by Ni-NTA agarose, but no change in purity.
Please advise if any one has done earlier
Thank you
T M P RAJU
If you have not already, you must make sure the protein is secreted. Not all cells secrete NGF even when overexpressed.
What does it mean 'no change in purity'? Are you sure you have enough protein secreted in medium or is it still inside of the cells? Make sure you are able to detect the protein on westernblot (I would recommend using HisTag antibody rather than Ni conjugate due to nonspecific binding of many other proteins).
If you have plenty of protein, Ni-NTA for purification is ok, but if not, another possibility is isolation on HisTag antibody which is bound to Protein G on agarose beads. Not very high capacity but very specific for all HisTagged proteins.
Firstly thank you all for your answers.
I have developed 4 novel chimeric fusion protein combinations and out of all Chimera 1 (smallest) has been successfully expressed in Sf9 insect cell system and purified to.
Yes, NGF and Chimera 1 has been successfully expressed as I have tested 6.5 microlitres of DIRECT media on western blot and clear band observed, but other chimeras aren't.
All buffers are at Ph 8.0. But now I have optimized purification using Talon (Cobalt, Ph 7.4) which is highly specific I found
But problem with rest of the chimeras which are expressed but not secreted into the media. I'm planning to purify these 3 chimeras from cells. I'm not sure How much I will gain from it
I welcome you all again for your valuable advise
I'm happy to discuss more in detail if any one of your in to similar field of work
Thank you
Raju
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