I'm having difficulty expressing and purifying the protein from my construct (Gateway cloning) in the pDest17 vector. The quantity is reduced after purification, with a significant background band.
I'm using lysis buffer with lysozyme and proteinase inhibitor overnight at 4oC and purifying with Ni-NTA beads, but the yield is low.
I'm using 10 mM imidazole for the wash buffer and 250 mM imidazole for the elution. I tried various IPTG concentrations and temperatures, but nothing worked. Even proteins are expressed without IPTG, and I am using BL21 cells. So, any advice on protein purification would be greatly appreciated.
In my hands, lysozyme is very inefficient at lysing intact E. coli cells, but works fairly well on partially lysed cells; thus, I would recommend something similar to what Erman suggested, having lysozyme present during sonication.
You can assay the efficiency of your lysis by running SDS-PAGE and comparing intact cells to your cells that have been through the lysing process. Spin both down (so cells / cell debris will be pelleted) and load the supernatant. As a positive control, use cells mixed with SDS loading buffer that have been boiled for 10 min. If the intensity of bands in the lysed lane is lower than the boiled lane, the lysis was not complete.
I would figure out the lysis problem first before trying more IPTG / temperature conditions, as none of these will yield better results if the cells are not being efficiently lysed.
I think one hour treatment with lysozyme (1mg/ml) will be sufficient followed by sonication and try to add 40mM imidazole when you are putting for binding ,which will enhance the binding affinity (in a competitive manner) of your protein with Ni-NTA beads.
Thank you all @Erman, @Aaron and @Chitti for your suggestions. But if the sonication facility is not available, what would be the best option then? Thanks
In lieu of sonication, the best method I can think of would be to use a French cell press, but these are probably even less commonly used than sonicators. Alternatively, assuming your protein is stable during the process, you could try subjecting the cell suspension to multiple freeze and thaw cycles. This takes much longer (mainly depending on the volume) but should still be effective, especially if lysozyme is present.
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