Dear colleague
does anyone know how to purify the secreted protein from leishmania into the superntant. I used leishmania as expression system for my protein and it should be in the superntant, I want to determine the protein by WB then purify it
can anyone help me in this issue or if there is a protocol from recent papers that can I fellow ?
Some things to consider in regard to purification of proteins from culture supernatant:
What else is going to be in the supernatant? Is it already rich in other proteins? Is it high in salts or other charged substances? Does it contain a large amount of high-molecular-weight substances? Does your expressed protein bear an affinity tag?
One way to start the purification would be to concentrate the medium using a pressurized ultrafiltration device. This will reduce the volume and concentrate the proteins, speeding up a subsequent capture step. If the medium is rich in proteins or other high-molecular-weight polymers, however, this may not be the best way to begin. If it is possible to grow the organism in a low-protein medium, it would be preferable.
You can collect the proteins from the medium by ammonium sulfate precipitation. This usually preserves the native structure of proteins, and it can also be used as an initial purification step because each protein precipitates at a different range of ammonium sulfate concentration. The precipitate can then be centrifuged to form a pellet, which is then resuspended in a small volume of buffer. The sample can then be dialyzed to lower the salt concentration and used for ion exchange chromatography, or it can be used directly for gel filtration chromatography.
If the culture medium has low ionic strength, you could capture the proteins by applying the medium directly to an ion exchange column, washing, and eluting with a salt gradient to begin the purification. If the volume of medium is very large, you could either begin with ultrafiltration to reduce the volume, or you could do batch binding by pouring the ion exchange resin into the medium, mixing it around for a while, then pouring it into a column for chromatography.
If the protein has been expressed with an affinity tag, you might be able to purify it, or partially purify it, by running the medium over the affinity resin and performing affinity chromatography. However, affinity chromatography may not be sufficient to achieve high purity, and it may be preferable to do some additional purification steps before or after, as already described. Ion exchange, gel filtration, and hydrophobic interaction chromatographies can all be applied in various orders to assist with purification.
You will require some method to follow the protein through the purification. If it is expressed at a very high level, you may be able to follow it simply by SDS-PAGE. If it is a very low-abundance protein and you have a specific antibody, you can follow it by Western blot or ELISA (ELISA is faster). If it is an enzyme for which you have a specific assay, you might be able to follow it using that assay.
Thanks Adam, actually my protein is fused to secreted acid phosphotase and its the only protein which secreted in the superntant and I did WB from the cell pellets it gave me positive result but I could not detect the protein from superntant by using WB and I used ultracenterfuage to concentrate the protein for WB. I just wounder how can I know if the protein separated from the vehicle protein (SAP) or not during the protein expression ?
Dear Dalia
I purpose you an appraoch as following scheme:
a-separation from non-protein components (lipids);
b- precipitation : in this case use the following steps
-do some differential precipitation with ammonium sulphate.
-For precipitation: dissolve ammonium sulphate into the medium in cycles to give first 20%, then 30%, 40% and higher saturation. After each step, remove by centrifugation the supernatant.
-resuspend each precipitate in an useful buffer for the next step.
c- Purfication: use of ionexchange chromatography/size fractionation or hydrophobic chromatography columns to further separate the target protein-containing fraction from the bulk protein.
Please find an interesting paper entitled: Purification of secreted protein from cell culture supernatant using Ni2+NTA chromatography. You can download the attached file.
Hoping to be helpful
Marius
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