Sorry I got confused with the wrong labeling. But the stoichiometry issue and poor expression is still the main problem. As Tom suggested, you should try to improve the expression. Poor expression often results in impurities copurifying. Also, if the interaction is not tight, why are you co-expressing? Unless coexpression is helping in some way (solubility ....) you are better off expressing (and purifying) them separately and then mixing them. often, when you co-express multiple proteins, you get poor expression as well.
Dhiraj ________________________________ From: CCP4 bulletin board <CCP4BB@JISCMAIL.AC.UK> on behalf of Tom Huxford <thuxf...@sdsu.edu> Sent: Tuesday, July 13, 2021 11:27 AM To: CCP4BB@JISCMAIL.AC.UK <CCP4BB@JISCMAIL.AC.UK> Subject: [External] Re: [ccp4bb] 60 kDa contamination in Rosetta cells I agree with Dhiraj' suggestion that the 60 kDa is not your biggest concern. Optimize soluble protein expression and pay attention to cell lysis. Is the sample remaining cold throughout the process? Are you using enough volume of buffer relative to solid cell pellet? Also, from the gel it does not look like your 37 and 15 kDa bands are co-eluting. And your lane labels in the figure are not right. Good news: this seems like a solvable problem. Keep after it! Tom Huxford. ============== Tom Huxford Structural Biochemistry Laboratory Department of Chemistry & Biochemistry San Diego State University (619) 594-1606 On Jul 13, 2021, at 5:22 AM, Dilip Badgujar <dilip....@gmail.com<mailto:dilip....@gmail.com>> wrote: Hello, Please find the attached gel picture for the reference and some additional information. Lysis buffer-50 mM Tris-pH 8.0, 300 mM NaCl, 1 mM DTT. 1X protease inhibitor cocktail. SEC column – Superdex 200. 16/300 One of the proteins is 37 kDa while the other one is15 kDa. I do not think that they are making that strong heterodimer. The total molecular of the complex would be ~ 52 kDa but now it is around 60 kDa. I am currently using lower temperature (16 ᵒC) but can try around 12 with low IPTG conc. Regards Dilip Badgujar On Mon, Jul 12, 2021 at 11:56 PM zaigham khan <mahmood.zaig...@gmail.com<mailto:mahmood.zaig...@gmail.com>> wrote: Hey Dilip, There are many reasons for this observation. Rafael is right, please do share the image of the gel. Also what are the exact sizes of the two proteins that you co-expressed? I have observed the heterodimeric and pentameric proteins on SDS-PAGE albeit the presence of DTT in the sample buffer, and despite boiling of the samples. Could this be that 60 KD is actually the hetero-dimer? One can perform western blotting, followed by the use of anti-polyhistidine and anti-Streptavidin antibodies on separate blots to confirm the suspected bands. Likewise SEC followed by WB may confirm the identity of the eluted proteins in different fractions after size exclusion chromatography. You may also cleave the tags, and then see the magic! Tom has correctly pointed out that induction at lower temperature is best achieved upon incubation of culture so that the temperature is dropped before the induction. Bon Voyage! -Z Zaigham M Khan, PhD Associate Scientist Icahn School of Medicine at Mount Sinai Department of Oncological Sciences 1470 Madison Avenue New York United States On Mon, Jul 12, 2021 at 2:45 AM Dilip Badgujar <dilip....@gmail.com<mailto:dilip....@gmail.com>> 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 -- Dilip C. Badgujar, (PhD) Post Doctoral Fellow, IIT Bomaby ________________________________ To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1 <Purification-Attempt1.png> ________________________________ 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/
