Dear all, I am posting a summary of How to avoid protein trafficking to Inclusion body???
My question was "What can be done to avoid a protein going inside inclusion body?The gene is cloned in pET30a with C-ter his tag and expressed in BL21-DE3 from 37 to 18C for 3-4 hr with .5mM of IPTG,it is going to inclusion body." Shivesh David Cobessi wrote, I do not know which kind of protein it is or the pET30a characteristics. But... You could first not add IPTG and see if the gene is expressed. It happens often. You could also decrease more the IPTG concentration. You could also clone the gene with the His-tag in Nter instead of Cter. You could also change the expression system. Good luck, David -- David Cobessi, PhD UMR7175-LC1, Institut Gilbert-Laustriat Ecole Superieure de Biotechnologie de Strasbourg Departement Recepteurs et Proteines Membranaires Boulevard Sebastien Brandt BP 10413 67412 Illkirch Cedex France Tel 33 (0)3 90 24 47 47 33 (0)6 08 16 43 40 Fax 33 (0)3 90 24 48 29 http://recepteurs.u-strasbg.fr http://site.voila.fr/d.cobessi Brenda Patterson wrote Lower temperature, use chaperones (e.g. TAKARA set), refolding? Mohinder Pal wrote I had the similar problems with the protein expression but I tried heat shock at 42 degree C for 25 mins before induction with IPTG and approximately 60% of the overexpressed protein become soluble. The explanation for this is the upregulation of heat shock proteins that may help in the refolding of the overexpressed proteins. So it is woth to try. Best of luck. Mohinder --------------------------------------------------------------------------------- Mohinder Pal Ph.D Student Protein Crystallography Group School of Biological Sciences University of Southampton Bassett Crescent East Southampton SO16 7PX UK [EMAIL PROTECTED] <mailto:[EMAIL PROTECTED]> Fax: +44 (0)23 8059 4459 Also worth trying a lower IPTG level ~ 0.1mM and or different media, e.g. TB, SOC or m9 instead of LB, it's all a bit random but sometimes these things can make a difference. Adding 3% EtOH on induction worked for me once, it's supposed to shock the cells and stimulate chaperone production, I was surprised I must say. Funny old world...... Giles Robertson D.Phil. [EMAIL PROTECTED] Tel: 02076316813 School of Crystallography, Birkbeck College, University of London, Malet Street, London, WC1E 7HX. - Peter J. Miller wrote On 2 Apr 2008, at 12:19, Brenda Patterson wrote: Lower temperature, use chaperones (e.g. TAKARA set), refolding? Quoting shivesh kumar <[EMAIL PROTECTED]>: Dear all, Sorry for the off-topic question... What can be done to avoid a protein going inside inclusion body.The gene is cloned in pET30a with C-ter his tag and expressed in BL21-DE3 from 37 to 18C for 3-4 hr with .5mM of IPTG,it is going to inclusion body.All suggestions are welcome. Thanx in advance. Shivesh Is there any reason that it has to be expressed solubly? Why not let it go into inclusion bodies and then refold it. That is what we do with most of our proteins...works great. Peter Peter J. Miller Collins Laboratory Department of Biochemistry and Biophysics University of North Carolina at Chapel Hill 919-966-9410 Louise Major wrote My favourite media is TPB - (paper is: Moore et al Protein expression and purification (1993) 4: 160-163). I've had luck with this and BL21 (DE3) Gold cells. I'm also a fan of cold shocking cells - after initial growth at 37 deg C, put flasks in an ice water bath for 10 min, then add IPTG and tranfer to 16-25 deg C for expression. This combination rescued a protein that behaved like ball bearings in all other conditions. It always seems worth trying an array of strains vs an array of media vs cold shock/heat shock/chaperones, and a bit of tweaking of [IPTG] Good luck, Lou - Show quoted text - At 11:06 02/04/2008, you wrote: Dear all, Sorry for the off-topic question... What can be done to avoid a protein going inside inclusion body.The gene is cloned in pET30a with C-ter his tag and expressed in BL21-DE3 from 37 to 18C for 3-4 hr with .5mM of IPTG,it is going to inclusion body.All suggestions are welcome. Thanx in advance. Shivesh ******************************************************** Louise Major Centre for Biomolecular Sciences North Haugh The University St Andrews Fife KY16 9ST Scotland Guentet wrote if you try to express a disulfide containing protein use Origami strains (Novagen) and keep temperature below 25 °C. Induction of chaperones as mentioned already is often very effective or co-expression of chaperones: de Marco, Deuerling et al. BMC Biotechnol. 2007; 7: 32. 2007 http://www.pubmedcentral.nih.gov/articlerender.fcgi?tool=pubmed&pubmedid=17565681 Godd luck, Guenter Priv.Doz.Dr. Guenter Fritz Fachbereich Biologie Sektion Naturwissenschaften Universitaet Konstanz http://www.biologie.uni-konstanz.de/fritz Universitaetsstrasse 10 Postfach M665 D-78457 Konstanz e-mail: [EMAIL PROTECTED] Tel. Office: +49-(0)7531 88 3205 Tel. Lab : +49-(0)7531 88 3687 Fax: +49-(0)7531 88 2966
