Maybe this has already been mentioned in which case apologies: I’d try rapid dilution. My only experience of having to refold worked both by dialysis and the dilution method but dilution had the advantage of allowing various conditions to be tested quickly and more controllably at small scale. I started with 100mgml-1 (1ml) protein in 5M GuHCl and did a 100 fold dilution with very rapid stirring. I followed this up years later by trying to refold using the Mosquito, tiny drops and a 96 well refolding screen. The plates set up set up as if they were crystallisation experiments and were sealed and imaged. The readout was visible precipitation in the drops. Those that stayed clear were perhaps the better conditions although I couldn’t categorically say they were; it was just a guide to follow up on. A better readout would be activity and the Mosquito could be replaced by a multichannel pipette. I guess it hinges on whether you are getting enough protein to start experimenting with.
Good luck! David From: CCP4 bulletin board [mailto:[email protected]] On Behalf Of Dipankar Manna Sent: 21 September 2017 11:12 To: [email protected] Subject: [ccp4bb] Precipitation issue during refolding Hi, I am working with a serine protease. As the protein is not soluble I am purifying it from the inclusion bodies followed by refolding. The protein shows good activity after refolding but the major concern is the 'precipitation'. Though the protein express quite well, but I loose almost 50-60% protein during refolding (Buffer: 100 mM Tris pH 8.0, 125 mM MgCl2, 100 mM KCL, 5% Glycerol, 2 mM GSH, 1 mM GSSG and 1 M NDSB) as it precipitates. Refolding is done in room temperature. I tried refolding at 4 degree, but it even end up with more precipitant. It also precipitates during concentrating, so in general I almost loose most of the protein during this refolding and concentration steps. I start with 6 lit culture that give around 1-2 mg protein in the final step, which I am not happy with. Any suggestion to deal this issue would be highly appreciated. Thank you in advance. Best, Dipankar -- Dipankar Manna, Ph.D Postdoctoral Researcher Department of Molecular Medicine Institute of Basic Medical Sciences University of Oslo, Domus Medica Oslo, Norway Mob : +47 451 66 517<tel:451%2066%20517> E-mail: [email protected]<mailto:[email protected]> [email protected]<mailto:[email protected]> http://www.med.uio.no/imb/english/people/aca/dipankam/<https://clicktime.symantec.com/a/1/Z0QRmWz8PLOsojkRzXM643ARr_3j2AFfOmMTGujbfy0=?d=EJUnC0JC140HvxIKhtldRVMAsaxyv9ByLkk9RuDiVEtBeWxpjQ8kpybIrM62kW347BbHXRwKoAr5A7XezOluJ5QCXbScS2PC2u2mvTzfP6Pq_cnJ4DCCn_dT_ulgWMwjFLxb2viV_FEZFqKHYlb1TthdRqoFSiMKVz-OPN7pf9fEZn9h_oDjsdpMDdFjv2EpM-YVSA1y6E9koNFvq-WXXT6-dbJVOQMfqgWp5Y4cIN-EIhIc_f7Zt-8NI4iy4ZkAEw4a8jVT97jmsrsetMcBp9JTYXDYVA4WqVVvh9m6G5VAwikNqWTXrHbIRNp7jAHh4XADV7qMgzo2KQKZSQG-bR4Ko5AwKNg6HrG9QZhkAbvBwphgaCq-g66N6sPhQqnwu_F4XRqfc_diTBOR12_KFLjmOAr2KaTKTz_dDlmhgM29&u=http%3A%2F%2Fwww.med.uio.no%2Fimb%2Fenglish%2Fpeople%2Faca%2Fdipankam%2F> ________________________________ AstraZeneca UK Limited is a company incorporated in England and Wales with registered number:03674842 and its registered office at 1 Francis Crick Avenue, Cambridge Biomedical Campus, Cambridge, CB2 0AA. This e-mail and its attachments are intended for the above named recipient only and may contain confidential and privileged information. If they have come to you in error, you must not copy or show them to anyone; instead, please reply to this e-mail, highlighting the error to the sender and then immediately delete the message. For information about how AstraZeneca UK Limited and its affiliates may process information, personal data and monitor communications, please see our privacy notice at www.astrazeneca.com<https://www.astrazeneca.com>
