Co expression of binding partner perhaps ? And then tagging the other protein partner ? Jürgen
...................... Jürgen Bosch Johns Hopkins Bloomberg School of Public Health Department of Biochemistry & Molecular Biology Johns Hopkins Malaria Research Institute 615 North Wolfe Street, W8708 Baltimore, MD 21205 Phone: +1-410-614-4742 Lab: +1-410-614-4894 Fax: +1-410-955-3655 http://lupo.jhsph.edu On Mar 6, 2014, at 2:13, "WENHE ZHONG" <wenhezhong.xmu....@gmail.com> wrote: > 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
