That is almost certainly GroEL. I would start by lowering expression temperature. Maybe try moving your warm cultures to room temperature (we use stir plates) as soon as you begin to see growth. Allow them a couple of hours to cool and then induce with low IPTG (0.1 mM) for 14-16 hours (overnight).
Tom Huxford. ============== Tom Huxford Structural Biochemistry Laboratory Department of Chemistry & Biochemistry San Diego State University (619) 594-1606 > On Jul 11, 2021, at 11:45 PM, Dilip Badgujar <[email protected]> wrote: > > Greetings, everyone > I am trying to co-express two mammalian proteins (less than 50 kDa MW) in > Rosetta cells but getting a contaminating band around 60 kDa. One of the > construct is in pET28a with His tag while the other is in pET21c with Strep > tag and I am adding all the three selection markers during growth of > pre-culture and during induction. Initially, cells were grown at 37 °C till > OD reaches to 0.6 then induced with 0.5mM IPTG and incubated at 16°C ON. When > I do purification using Streptactin resin; I can see proteins of my interest > bound to the resin along with contaminating protein at 60 kDa. I have tried > performing size exclusion as a follow up step but they are co-eluting in void > volume. I have also tried to wash with MgCL2-ATP solution but it co-elution > with contaminant. I am looking for valuable suggestions to avoid the > contamination during or after expression. > > Thanks in advance. > > Regards > > Dilip > > > 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/
