Hi there,

I am working with a GST tagged recombinant protein with a Mw of 35 kDa and pI of 9.0. The protein is expressed well in E.coli to a desired level and available in the supernatant after cell lysis. I am using a pre-clarification step with 500 kDa hollow fibre module to remove some impurities before I go for affinity purification. I am using 50 mM tris buffer, pH 8.0 containing 150 mM NaCl for lysis and diafiltrating buffer. Unfortunately, the desired protein end up in the retentate instead of permeate.

Can someone help to overcome this issue?

I am planning for the below experiments to troubleshoot the problem:

1. Increase salt concentration to 1 M or even higher

2. Decrease the buffer pH to 7.4 instead of 8.0 as it was close to pI of the protein

3. Any previous observations of temporary self-assembling GST tagged proteins under native conditions?. When analysed under reduced conditions, it is running as a monomer.

Thanks in advance.