Dear Jack, You may want to consider using a Strep2 tag (instead of your tag or additional to it) as it contains one Trp. I used it for some of my smaller proteins (without natural Trp), which works fine for detection and purification. Typically, I would go for N-terminal His and C-terminal Strep2, so you can be sure to purify homogenous FL proteins.
Cheers, Jonas -- Jonas Weidenhausen PhD Student AG Sinning BZH Heidelberg University, room: 524 INF 328, 69120 Heidelberg Phone: +49 6221 54-4786 jonas.weidenhau...@bzh.uni-heidelberg.de<mailto:jonas.weidenhau...@bzh.uni-heidelberg.de> On 19. Feb 2022, at 19:42, Tanner, John J. <tanne...@missouri.edu<mailto:tanne...@missouri.edu>> wrote: Casper: You make a good point about using 205 nm. I checked our system this morning and the wavelength is fixed at 280 nm (U9-L). Artem: Did you consider secondary structure and/or amino acid residue type when choosing sites for mutagenesis? Is your work published? From: CCP4 bulletin board <CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK>> on behalf of Casper Wilkens <00005d34ab0aef35-dmarc-requ...@jiscmail.ac.uk<mailto:00005d34ab0aef35-dmarc-requ...@jiscmail.ac.uk>> Date: Saturday, February 19, 2022 at 2:19 AM To: CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK> <CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK>> Subject: [ccp4bb] Sv: [ccp4bb] Strategies for increasing Trp content of proteins WARNING: This message has originated from an External Source. This may be a phishing expedition that can result in unauthorized access to our IT System. Please use proper judgment and caution when opening attachments, clicking links, or responding to this email. Is absorbance at 205 nm not a possibility, Jack? Casper Wilkens Asst. Prof. Structural Enzymology & Biorefining DTU Bioengineering Technical University of Denmark [http://www.dtu.dk/%7E/media/DTU_Generelt/Andet/DTU_email_logo_01.gif] Department of Biotechnology and Biomedicine Søltofts Plads Building 224 Room 028 2800 Kgs. Lyngby c...@dtu.dk<mailto:c...@bio.dtu.dk> www.bioengineering.dtu.dk/<https://nam02.safelinks.protection.outlook.com/?url=http%3A%2F%2Fwww.bioengineering.dtu.dk%2F&data=04%7C01%7Ctannerjj%40MISSOURI.EDU%7Ccd1aefc8cc284ea9e44d08d9f38074db%7Ce3fefdbef7e9401ba51a355e01b05a89%7C0%7C0%7C637808555459123981%7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C3000&sdata=viaDJTf6EgVVTGxk4AGAWEyluluwh9rtnwUCUUULmo0%3D&reserved=0> https://www.cazypedia.org/index.php/User:Casper_Wilkens<https://nam02.safelinks.protection.outlook.com/?url=https%3A%2F%2Fwww.cazypedia.org%2Findex.php%2FUser%3ACasper_Wilkens&data=04%7C01%7Ctannerjj%40MISSOURI.EDU%7Ccd1aefc8cc284ea9e44d08d9f38074db%7Ce3fefdbef7e9401ba51a355e01b05a89%7C0%7C0%7C637808555459123981%7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C3000&sdata=yJVCEDisnY3LmyvCPPjFKmybTNFrQHlEKjrEmQPVtHE%3D&reserved=0> https://twitter.com/protein_artist<https://nam02.safelinks.protection.outlook.com/?url=https%3A%2F%2Ftwitter.com%2Fprotein_artist&data=04%7C01%7Ctannerjj%40MISSOURI.EDU%7Ccd1aefc8cc284ea9e44d08d9f38074db%7Ce3fefdbef7e9401ba51a355e01b05a89%7C0%7C0%7C637808555459123981%7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C3000&sdata=bKeX%2FhUL7pnDA3DxL5ZFcTUlHkL5x2Vd7VsrJNPA0H0%3D&reserved=0> https://www.researchgate.net/profile/Casper_Wilkens<https://nam02.safelinks.protection.outlook.com/?url=https%3A%2F%2Fwww.researchgate.net%2Fprofile%2FCasper_Wilkens&data=04%7C01%7Ctannerjj%40MISSOURI.EDU%7Ccd1aefc8cc284ea9e44d08d9f38074db%7Ce3fefdbef7e9401ba51a355e01b05a89%7C0%7C0%7C637808555459123981%7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C3000&sdata=VuBOXy%2BOSmA6usq%2BahiVlN2%2Fm19%2FnlVEqNXDRMRnrkY%3D&reserved=0> ________________________________ Fra: CCP4 bulletin board <CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK>> på vegne af Artem Evdokimov <artem.evdoki...@gmail.com<mailto:artem.evdoki...@gmail.com>> Sendt: 19. februar 2022 02:12:50 Til: CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK> Emne: Re: [ccp4bb] Strategies for increasing Trp content of proteins We have had success placing single trp on the outside of proteins, as weird as it sounds it works pretty well. If your protein is easy to express you can try a few choices. If your protein is difficult to express, a fusion with a trp-positive domain or with gfp may be a better option. There are many other options but their application is best considered with more information in hand Artem On Fri, Feb 18, 2022, 8:02 PM Tanner, John J. <tanne...@missouri.edu<mailto:tanne...@missouri.edu>> wrote: Dear CCP4BB, We are working on a protein that has no Trp residues, which makes chromatography challenging due to the low absorbance at 280 nm (Abs 0.1% = 0.104). Does anyone have experience using mutagenesis to increase the Trp content of proteins? Thanks, Jack Tanner -- John J. Tanner Professor of Biochemistry and Chemistry Associate Chair of Biochemistry Department of Biochemistry University of Missouri 117 Schweitzer Hall 503 S College Avenue Columbia, MO 65211 Phone: 573-884-1280 Email: tanne...@missouri.edu<mailto:tanne...@missouri.edu> https://cafnrfaculty.missouri.edu/tannerlab/ Lab: Schlundt Annex rooms 3,6,9, 203B, 203C Office: Schlundt Annex 203A ________________________________ 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://nam02.safelinks.protection.outlook.com/?url=https%3A%2F%2Fwww.jiscmail.ac.uk%2Fcgi-bin%2FWA-JISC.exe%3FSUBED1%3DCCP4BB%26A%3D1&data=04%7C01%7Ctannerjj%40MISSOURI.EDU%7Ccd1aefc8cc284ea9e44d08d9f38074db%7Ce3fefdbef7e9401ba51a355e01b05a89%7C0%7C0%7C637808555459123981%7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C3000&sdata=lqUvJHHOWqoIPD4zzPwhjINYcanSB1R4e97nPfeBiSA%3D&reserved=0> ________________________________ 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://nam02.safelinks.protection.outlook.com/?url=https%3A%2F%2Fwww.jiscmail.ac.uk%2Fcgi-bin%2FWA-JISC.exe%3FSUBED1%3DCCP4BB%26A%3D1&data=04%7C01%7Ctannerjj%40MISSOURI.EDU%7Ccd1aefc8cc284ea9e44d08d9f38074db%7Ce3fefdbef7e9401ba51a355e01b05a89%7C0%7C0%7C637808555459123981%7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C3000&sdata=lqUvJHHOWqoIPD4zzPwhjINYcanSB1R4e97nPfeBiSA%3D&reserved=0> ________________________________ 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://nam02.safelinks.protection.outlook.com/?url=https%3A%2F%2Fwww.jiscmail.ac.uk%2Fcgi-bin%2FWA-JISC.exe%3FSUBED1%3DCCP4BB%26A%3D1&data=04%7C01%7Ctannerjj%40MISSOURI.EDU%7Ccd1aefc8cc284ea9e44d08d9f38074db%7Ce3fefdbef7e9401ba51a355e01b05a89%7C0%7C0%7C637808555459123981%7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C3000&sdata=lqUvJHHOWqoIPD4zzPwhjINYcanSB1R4e97nPfeBiSA%3D&reserved=0> ________________________________ 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/
