protein purification from media supernatant? - protein purification of ECM proteins (Dec/18/2008 )
Hello everyone,
I am working on an excreted, extracellular matrix protein. I have the vector with appropriate sequences (promoters ect), it is a pcDNA3.1. I have a line that over-expresses the protein of interest (checked by western blot). The protein contains both a 6x his tag and a V5 tag (the his for purification and the V5 for detection). I now need to purify the protein. I cannot use bacterial expression system because I need the active protein and post-translational modifications are extremely important for my protein. I therefore have to use the mammalian transfected cells and take the supernatant to do it (as the protein that is made inside the cell is very little compared to outside the cells).
I have the quiagen's Ni-NTA magnetic agarose beads system. And there is protocol that describe using mammalian cells, but the protocol is for cell lysates. Does anybody have experience extracting a protein from the supernatant of mammalian cells that is his-tagged? Any ideas suggestions protocols will be more than useful at this point. Do I need to change the buffer, if so what methods/companies you suggest? I am asking that because in the media there is lots of glutamine and glucose and serum which reduce Ni and therefore reducing its binding capacity.
Thanks a lot in advance for the help! 
I am working on an excreted, extracellular matrix protein. I have the vector with appropriate sequences (promoters ect), it is a pcDNA3.1. I have a line that over-expresses the protein of interest (checked by western blot). The protein contains both a 6x his tag and a V5 tag (the his for purification and the V5 for detection). I now need to purify the protein. I cannot use bacterial expression system because I need the active protein and post-translational modifications are extremely important for my protein. I therefore have to use the mammalian transfected cells and take the supernatant to do it (as the protein that is made inside the cell is very little compared to outside the cells).
I have the quiagen's Ni-NTA magnetic agarose beads system. And there is protocol that describe using mammalian cells, but the protocol is for cell lysates. Does anybody have experience extracting a protein from the supernatant of mammalian cells that is his-tagged? Any ideas suggestions protocols will be more than useful at this point. Do I need to change the buffer, if so what methods/companies you suggest? I am asking that because in the media there is lots of glutamine and glucose and serum which reduce Ni and therefore reducing its binding capacity.
Thanks a lot in advance for the help!
There are a few options. One that worked very well for me when I was purifying from TC medium was transverse flow filtration (TFF) to get rid of the serum components and small molecules and to reduce the volume followed by diafiltration to complete the buffer exchange.
If you don't have that gear, you could do some differential precipitation with ammonium sulphate. Google the protocol, but basically you dissolve ammonium sulphate into the medium in cycles to give first 20%, then 30%, 40% and higher saturation. Different proteins will (reversibly) precipitate at each stage, which are removed by centrifugation. Once the precipitate is resuspended in a useful buffer for the next step, the proteins go back into solution. The resuspended protein mixes are then run on SDS-PAGE to see which fraction has your protein. You get good preliminary purification, and remove the medium's contaminating compounds.
Thanks for your reply that is very useful- I've never tried this protocol before. I hope it will work for me.