Hi Herman, We learned a few tricks of using MBP fusion for solving an interesting fold of a receptor extracellular domain from our recent publication (https://pubmed.ncbi.nlm.nih.gov/32541044/ <https://pubmed.ncbi.nlm.nih.gov/32541044/>). (1) the length of a linker between MBP and protein of interest is critical. Use a computational modeling approach to figure out a reasonable linker, if possible. (2) take advantage of engineered MBP e.g., surface entropy reduction mutations and ion-mediated dimerization mutations (a good review https://pubmed.ncbi.nlm.nih.gov/26682969/ <https://pubmed.ncbi.nlm.nih.gov/26682969/>; https://pubmed.ncbi.nlm.nih.gov/26850170/ <https://pubmed.ncbi.nlm.nih.gov/26850170/>) that do help. (3) use either E. coli Shuffle cells or mammalian cells to deal with the disulfide bonds and characterize the protein folding e.g. CD or SEC (not ideal, but simple).
Hope this helps. Best wishes, Tao-Hsin Tao-Hsin Chang, DPhil Research Specialist Howard Hughes Medical Institute > On Mar 15, 2021, at 5:07 AM, Schreuder, Herman /DE > <herman.schreu...@sanofi.com> wrote: > > Dear Bulletin Board, > Sorry for the slightly off-topic question, but we are struggling with a > receptor domain that expresses well as a fusion protein, but gets lost the > moment it is cleaved from the fusion partner. It could be that the receptor > domain is not or misfolded, but it could also be a solubility problem. > > I have seen some crystal structures of fusion proteins with MBP and for > membrane proteins, T4-lysozyme fusions are often crystallized. What is your > experience? Would it be worth trying to express and crystallize a fusion > protein, or would it be better to look for other constructs, e.g. to include > more receptor domains? > > Thank you very much for your advice! > Herman > > > > > To unsubscribe from the CCP4BB list, click the following link: > https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1 > <https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1> ######################################################################## To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1 This message was issued to members of www.jiscmail.ac.uk/CCP4BB, a mailing list hosted by www.jiscmail.ac.uk, terms & conditions are available at https://www.jiscmail.ac.uk/policyandsecurity/
