Dear CCP4 friends, I have been searching around the CCP4 mails for the discussion of soluble protein aggregations, as I have the same problem recently. Many efforts I have tried but without success. Here, I would like to summarise what I have done and maybe any of you come across some ideas.
1. My protein (not membrane protein) is around 35kDa with a pI of 8.6. It was expressed in E.coli as a fusion protein where its N-ter was fused by MBP-tag with a flexible linker (Factor Xa cleavage site available). The whole fusion protein is ~77kDa (pI 6.1). 2. After MBPTrap affinity purification, the protein was run through gel-filtration column it came out at void volume. The aggregation was also confirmed by DLS. The buffer condition is: 20 mM HEPES, pH7.4, 125 mM KCl, 20% glycerol, 40 mM maltose, 5 mM DTT. 3. I made several truncations and only one gave me a monomer (The other truncations were all soluble aggregates). However, this monomeric truncation is small (~5 kDa) and not important for the function (not interesting fragment). At least, this monomeric truncation suggests that the aggregation is not due to the aggregation of MBP. The truncations were designed in both two versions: long linker (including TEV cleavage site) and short linker (-Ser-Ser-Ser-). 4. Hampton detergent screens and high salt (1-2 M KCl or NaCl) were tried on both truncations and full-length protein, but none of them work (DLS confirm). Divalents and other ions should not bind to this protein. 5. The full-length aggregated protein is still functional in Gel-shift assay, which indicates the protein is corrected folding. I guess "soluble aggregation" is a common problem for tough proteins. If anyone has experience on it and can share with us, please drop an email. Kind regards, Wenhe
