Hi Dipankar, Are you expressing the active protease, or the inactive precursor (zymogen)? Although we never systematically looked at it, I strongly suspect that many active proteases destroy the expression host and you will therefor only find clones expressing the protein in inclusion bodies.
Upon refolding, autoproteolysis may become a problem. Since it is a bimolecular process, the rate goes up with the square of the concentration. So during the concentration steps, you may have to add a strong inhibitor of your protease. If it is a new structure, you could consider adding an irreversible, covalent inhibitor to completely inhibit your protease. PPACK has been used for this in a large number of proteases, but it depends on the substrate specificity of your protease. Alternative, you could mutate the active site serine into an alanine. So in summary: -produce the protease as a zymogen and activate afterwards. There is a reason nature produces most proteases as zymogens. -make sure you prevent autolysis during refolding and especially during concentration. Good luck! Herman Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Dipankar Manna Gesendet: Donnerstag, 21. September 2017 12:12 An: CCP4BB@JISCMAIL.AC.UK Betreff: [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: dipankar.ma...@medisin.uio.no<mailto:dipankar.ma...@kjemi.uio.no> dipankar.biot...@gmail.com<mailto:dipankar.biot...@gmail.com> http://www.med.uio.no/imb/english/people/aca/dipankam/<https://urldefense.proofpoint.com/v2/url?u=http-3A__www.med.uio.no_imb_english_people_aca_dipankam_&d=DwMFaQ&c=Dbf9zoswcQ-CRvvI7VX5j3HvibIuT3ZiarcKl5qtMPo&r=HK-CY_tL8CLLA93vdywyu3qI70R4H8oHzZyRHMQu1AQ&m=HSRNC9Jh2XZ2Ly50p2YjlU6bSE_DZSK-sJPW0R9pwoY&s=PZuIYH74QBag5tHOrebvRFBO6zLO2DHBkp7g4f0cwr0&e=>
