Hi Akila, I'm curious about your choice of pET22b. If you cut out the signal peptide that comes with the vector and express your protein in the cytoplasm, then what I am about to say doesn't apply. However, if you are exporting the protein into the periplasm, have you considered doing osmotic shock instead of lysing the cells? When you lyse the cells, you are likely to get misfolded/unprocessed protein from the cytoplasm that will likely precipitate. Pun
On Wed, Mar 29, 2017 at 8:38 PM, Akilandeswari Gopalan < akilaibt2...@gmail.com> wrote: > Dear all, > I am a PhD student doing structural studies on a few proteins from > Mycobacterium tuberculosis. The gene encoding the proteins I work on are > cloned into pet22b with c terminal His tag. the proteins are expressing > well. upon purification I am getting good yield of protein but during > dialysis, the proteins precipitate. Kindly suggest some solutions to avoid > aggregation. pI of one protein is 9.7 and that of the other is 5.6 > I am using 25mM Tris pH 7.5 and 100 mM NaCl buffer with 5mM > beta-mercaptoethanol and 0.5% triton x 100 for lysis, the same buffer with > 20-30mM imidazole for washing and 300mM imidazole for eluting the proteins. > > Thank you > Regards > Akila > > -- > Akilandeswari G > >
