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:[email protected] <mailto:[email protected]> 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:[email protected]] Im Auftrag von Claudia Scotti Gesendet: Mittwoch, 10. März 2010 12:46 An: [email protected] 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>
