Re: [ccp4bb] Off-topic - Domain termini for construct design
Hi - I do not think there are rules. There is a logic, to never truncate within secondary structure elements, and to prefer to delete regions that have a high probability to be disordered. With excuses for the non-ccp4 plugin, you can use our web server to help you design these. http://xtal.nki.nl/ccd/Welcome.html A. On Mar 10, 2010, at 12:45, Claudia Scotti wrote: Dear All, I'd like to espress a protein for crystallisation as a whole, but also to try separate expression of its two domains. Although prediciting domain boundaires is not difficult, I know that it is worthwhile testing several constructs to improve espression in soluble form. In several works I've seen that the choice is to keep or give up some aminoacids (for example 4 or 5) at the N and/or C- termini and to test them to see which expresses best. I was wondering if there are defined rules to choose these variants and/or to modify the N and C termini of a protein of interest when it is not important to keep them true (=native) in order to optimise expression levels. Is there any good reference about this, please? Personal experiences are welcome. Many thanks, Claudia Claudia Scotti Dipartimento di Medicina Sperimentale Sezione di Patologia Generale Universita' di Pavia Piazza Botta, 10 27100 Pavia Italia Tel. 0039 0382 986335/8/1 Facs 0039 0382 303673 Hotmail: Free, trusted and rich email service. Get it now. P please don't print this e-mail unless you really need to Anastassis (Tassos) Perrakis, Principal Investigator / Staff Member Department of Biochemistry (B8) Netherlands Cancer Institute, Dept. B8, 1066 CX Amsterdam, The Netherlands Tel: +31 20 512 1951 Fax: +31 20 512 1954 Mobile / SMS: +31 6 28 597791
Re: [ccp4bb] Off-topic - Domain termini for construct design
Hello Claudia, have you thought of, instead of rules, using restricted proteolysis combined with N-terminal sequencing in order to better estimate a good position at least for the N-terminal part of your protein? Tim On Thu, Mar 11, 2010 at 09:26:53AM +0100, Anastassis Perrakis wrote: Hi - I do not think there are rules. There is a logic, to never truncate within secondary structure elements, and to prefer to delete regions that have a high probability to be disordered. With excuses for the non-ccp4 plugin, you can use our web server to help you design these. http://xtal.nki.nl/ccd/Welcome.html A. On Mar 10, 2010, at 12:45, Claudia Scotti wrote: Dear All, I'd like to espress a protein for crystallisation as a whole, but also to try separate expression of its two domains. Although prediciting domain boundaires is not difficult, I know that it is worthwhile testing several constructs to improve espression in soluble form. In several works I've seen that the choice is to keep or give up some aminoacids (for example 4 or 5) at the N and/or C-termini and to test them to see which expresses best. I was wondering if there are defined rules to choose these variants and/or to modify the N and C termini of a protein of interest when it is not important to keep them true (=native) in order to optimise expression levels. Is there any good reference about this, please? Personal experiences are welcome. Many thanks, Claudia Claudia Scotti Dipartimento di Medicina Sperimentale Sezione di Patologia Generale Universita' di Pavia Piazza Botta, 10 27100 Pavia Italia Tel. 0039 0382 986335/8/1 Facs 0039 0382 303673 Hotmail: Free, trusted and rich email service. Get it now. P please don't print this e-mail unless you really need to Anastassis (Tassos) Perrakis, Principal Investigator / Staff Member Department of Biochemistry (B8) Netherlands Cancer Institute, Dept. B8, 1066 CX Amsterdam, The Netherlands Tel: +31 20 512 1951 Fax: +31 20 512 1954 Mobile / SMS: +31 6 28 597791 -- -- Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A signature.asc Description: Digital signature
[ccp4bb] AW: [ccp4bb] Off-topic - Domain termini for construct design
Dear Claudia, Though not a fixed rule, here are a few general things that we consider: - N- and C-termini of homologs (eg from BLAST) - Secondary structure predictions - Disorder prediction (eg disembl or grooms servers) - Limited proteolysis to experimentally detect floppy ends Try to accumulate as much information as possible to guide your construct design. In case of limited proteolysis: sometimes the truncated pieces are too short to be revealed on an SDS-PAGE. You may need mass spec to reveal cutting. Mit freundlichen Grüßen / Kind regards, Dr. Alexander Pautsch Boehringer Ingelheim Pharma GmbH Co. KG A Leitstrukturfindung Tel.: +49 (7351) 54-4683 Fax: +49 (7351) 54-97924 mailto:alexander.paut...@boehringer-ingelheim.com mailto:alexander.paut...@boehringer-ingelheim.com Boehringer Ingelheim Pharma GmbH Co. KG, Sitz: Ingelheim am Rhein; Registergericht Mainz: HR A 22206; Komplementär Boehringer Ingelheim Deutschland GmbH; Geschäftsführung: Dr. Engelbert Günster (Vorsitzender), Ralf Gorniak, Felix Gutsche, Mark Hagmann, Dr. Martin Wanning; Vorsitzender des Aufsichtsrates: Engelbert Tjeenk Willink; Sitz: Ingelheim am Rhein; Registergericht Mainz: HR B 23260 Diese E-Mail ist vertraulich zu behandeln. Sie kann besonderem rechtlichen Schutz unterliegen. Wenn Sie nicht der richtige Adressat sind, senden Sie bitte diese E-Mail an den Absender zurück, löschen die eingegangene E-Mail und geben den Inhalt der E-Mail nicht weiter. Jegliche unbefugte Bearbeitung, Nutzung, Vervielfältigung oder Verbreitung ist verboten. / This e-mail is confidential and may also be legally privileged. If you are not the intended recipient please reply to sender, delete the e-mail and do not disclose its contents to any person. Any unauthorized review, use, disclosure, copying or distribution is strictly prohibited. Von: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] Im Auftrag von Claudia Scotti Gesendet: Mittwoch, 10. März 2010 12:46 An: CCP4BB@JISCMAIL.AC.UK Betreff: [ccp4bb] Off-topic - Domain termini for construct design Dear All, I'd like to espress a protein for crystallisation as a whole, but also to try separate expression of its two domains. Although prediciting domain boundaires is not difficult, I know that it is worthwhile testing several constructs to improve espression in soluble form. In several works I've seen that the choice is to keep or give up some aminoacids (for example 4 or 5) at the N and/or C-termini and to test them to see which expresses best. I was wondering if there are defined rules to choose these variants and/or to modify the N and C termini of a protein of interest when it is not important to keep them true (=native) in order to optimise expression levels. Is there any good reference about this, please? Personal experiences are welcome. Many thanks, Claudia Claudia Scotti Dipartimento di Medicina Sperimentale Sezione di Patologia Generale Universita' di Pavia Piazza Botta, 10 27100 Pavia Italia Tel. 0039 0382 986335/8/1 Facs 0039 0382 303673 Hotmail: Free, trusted and rich email service. Get it now. https://signup.live.com/signup.aspx?id=60969
[ccp4bb] Off-topic - Domain termini for construct design
Dear All, I'd like to espress a protein for crystallisation as a whole, but also to try separate expression of its two domains. Although prediciting domain boundaires is not difficult, I know that it is worthwhile testing several constructs to improve espression in soluble form. In several works I've seen that the choice is to keep or give up some aminoacids (for example 4 or 5) at the N and/or C-termini and to test them to see which expresses best. I was wondering if there are defined rules to choose these variants and/or to modify the N and C termini of a protein of interest when it is not important to keep them true (=native) in order to optimise expression levels. Is there any good reference about this, please? Personal experiences are welcome. Many thanks, Claudia Claudia Scotti Dipartimento di Medicina Sperimentale Sezione di Patologia Generale Universita' di Pavia Piazza Botta, 10 27100 Pavia Italia Tel. 0039 0382 986335/8/1 Facs 0039 0382 303673 _ Hotmail: Free, trusted and rich email service. https://signup.live.com/signup.aspx?id=60969
Re: [ccp4bb] Off-topic - Domain termini for construct design
Dear Claudia, This could be a good starting point: Protein Expr Purif. 2008 Apr;58(2):210-21. Epub 2007 Nov 22. The use of systematic N- and C-terminal deletions to promote production and structural studies of recombinant proteins. Gräslund S, Sagemark J, Berglund H, Dahlgren LG, Flores A, Hammarström M, Johansson I, Kotenyova T, Nilsson M, Nordlund P, Weigelt J. PMID: 18171622 [PubMed - indexed for MEDLINE] HTH, Luca On Mar 10, 2010, at 12:45 PM, Claudia Scotti wrote: Dear All, I'd like to espress a protein for crystallisation as a whole, but also to try separate expression of its two domains. Although prediciting domain boundaires is not difficult, I know that it is worthwhile testing several constructs to improve espression in soluble form. In several works I've seen that the choice is to keep or give up some aminoacids (for example 4 or 5) at the N and/or C-termini and to test them to see which expresses best. I was wondering if there are defined rules to choose these variants and/or to modify the N and C termini of a protein of interest when it is not important to keep them true (=native) in order to optimise expression levels. Is there any good reference about this, please? Personal experiences are welcome. Many thanks, Claudia Claudia Scotti Dipartimento di Medicina Sperimentale Sezione di Patologia Generale Universita' di Pavia Piazza Botta, 10 27100 Pavia Italia Tel. 0039 0382 986335/8/1 Facs 0039 0382 303673 Hotmail: Free, trusted and rich email service. Get it now.
