I am now working with a membrane protein, which is a 50KDa transporter with 12 trans-membrane-helixes.
The protein is produced heterologously in E.coli with a C-terminal His-tag. I purified the protein with Ni-NTA beads and then gel filtration. Everything was fine when I purified it in a small scale, like 4L culture. However, when I was trying to upscale the process (purify protein from16L culture), the protein started aggregating, judging by gel filtration profile. I am attaching the two profiles to make this more clear.
Does anyone know what happened? And what can I do to improve this?
Thanks so much!