Hi Herman I agree with all discussed already.
First, is your protein of interest actually folded or just solubilized by the large fusion partner? If it is folded, worth considering crystallization of the fusion protein, as a “desperate measure” approach. The key issue will be making the linkage between the proteins reasonably rigid, to increase chances of crystallization. In GPCRs, T4L is usually inserted into a loop, which obviously will constrain it more than just one linkage at the terminus. There is quite a bit of literature on the topic already, and some nice tools like the MBP with increased crystallization propensity, as already pointed out. Let me know if I can help finding any key papers. But at the end of the day, my feeling is people try this a lot, as setting up some crystallization plates is easier than troubleshooting cleavage, purification and making and purifying new constructs. Despite this, other than specific cases like GPCRs, there are not that many successful examples really. So my feeling is this approach doesn’t work that often without a lot of trial and optimization. Best wishes Bostjan -- Bostjan Kobe FAA Australian Research Council Laureate Fellow Professor of Structural Biology School of Chemistry and Molecular Biosciences and Institute for Molecular Bioscience (Division of Chemistry and Structural Biology) and Centre for Infectious Disease Research Cooper Road University of Queensland Brisbane, Queensland 4072 Australia Phone: +61 7 3365 2132 Fax: +61 7 3365 4699 E-mail: b.k...@uq.edu.au<applewebdata://A220FB9B-AAC1-4876-9D6D-5C9F47D3C087/b.k...@uq.edu.au> URL: http://www.scmb.uq.edu.au/staff/bostjan-kobe Office: Building 76 Room 329 Notice: If you receive this e-mail by mistake, please notify me, and do not make any use of its contents. I do not waive any privilege, confidentiality or copyright associated with it. Unless stated otherwise, this e-mail represents only the views of the Sender and not the views of The University of Queensland. From: CCP4 bulletin board <CCP4BB@JISCMAIL.AC.UK> on behalf of Tao-Hsin Chang <taohsin.ch...@gmail.com> Reply to: Tao-Hsin Chang <taohsin.ch...@gmail.com> Date: Tuesday, 16 March 2021 at 6:55 am To: "CCP4BB@JISCMAIL.AC.UK" <CCP4BB@JISCMAIL.AC.UK> Subject: Re: [ccp4bb] crystallizing fusion proteins 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/). (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/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<mailto: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 ________________________________ To unsubscribe from the CCP4BB list, click the following link: 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/
