[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.
[ccp4bb] PhD position in protein crystallography
PhD position in crystallization and structure solution of natural and appropriate variants of the sponge protein silicatein and nanobiotechnology applications TOPIC: Research in the field of biomineralization and applications in nanobiotechnology. Biomineralization is the formation of minerals by living cells and organisms. To understand the processes involved in biomineralization (at the cutting edge between inorganic and organic world)and for nanobiotechnology applications, the structure determination of natural and appropriate variants of silicatein from marine demosponge Suberites domuncula is required. The prospective student will carry out European funded research (Marie Curie ITN network BIOMINTEC) in two of the partner institutions, NCSR Demokritos in Greece and Johannes Gutenberg-Universitaet, Mainz in Germany and will operate within a network of institutions highly specialized in biomineralisation. The project includes participation in workshops and summer schools. Motivation to travel and adapt to a different country and to integrate efficiently in a new working team is fundamental. TYPE OF APPOINTMENT-DURATION-STARTING DATE: Full-time contracts in the two institutions (overall period 36 months), starting April 2010 at NCSR Demokritos. PROFILE-ELIGIBILITY Applicants should have a BS or MSc degree in chemistry, biological sciences or related sciences and £4 years research experience at the time of hiring. They can be nationals of European Union or Associated Countries, other than the country of the host organisations (Greece Germany). Candidates from countries outside EU are also eligible. Greek or German citizens who have resided in Greece or Germany for less than 12 months in the 3 years prior to the hiring dates are also eligible. BENEFITS The successful candidate as a Marie Curie fellow will enjoy high salary (according to the Marie Curie guidelines with social security benefits) and additionally a mobility allowance covering family obligations, travel allowance and career exploratory allowance. HOW TO APPLY Applicants should forward electronically (a) their detailed CV, (b) copies of published articles, (c) names, e-mails/phone numbers of two referees to: Dr. I. M. Mavridis Institute of Physical Chemistry National Center for Scientific Research Demokritos Aghia Paraskevi 1520, Athens -Greece mavr...@chem.demokritos.gr
Re: [ccp4bb] Reg Protein purification
Dear Sivaraman I worked on some protein with DNA contamination recently. Adding DNase and increasing IMAC wash volume at the same time changed 280/260 ratio and yield reproducible protein crystals, not high resolution yetIn case your protein can't stand other treatment... (Sorry Tommi, I hit the wrong reply button.) Best, Zheng (Joe) Zhou On Sat, Mar 6, 2010 at 8:23 PM, Sivaraman Padavattan s.padavat...@gmail.com wrote: Dear All, We are trying to purify an enzyme, which requires the co-factor NAD+ during catalysis by affinity column (Ni-NTA). After induction, the bacterial cells were harvested and lysed with 20 mM Tris pH 7.2, 500 mM NaCl, 5% glycerol, 5 MM B-ME. The resultant supernatant was passed through Ni-NTA and bound protein eluted with increasing concentration of Imidazole. The eluted proteins was concentrated and load onto gelfiltration (Superdex S-75 16/60) column. Our protein eluted as a aggregate along with other protein, where A260 was much greater than A280, indicative of large fraction of nucleic acid contamination. The eluant also appeared as a smear on 1% agarose gel electrophoresis. We introduced 1M NaCl in the lysis buffer to prevent the nucleic acid interaction. But most of our protein went in pellet after cell lysis. We look forward to your valuable suggestion to purify the protein free of nucleic acid contamination. Thanks in advance, Sivaraman Padavattan
[ccp4bb] MAHORI
Dear All, I have created this website MAHORI (http://felix.bioc.cam.ac.uk) to display ligand interaction with proteins in the PDB. There is a lot of room for improvement and I really hope to get feedback from the CCP4 community so that I can make it more useful to chemical biology, medicinal chemistry, fragment design research. Cheers, Duangrudee
[ccp4bb] crystallization plates and robotics
Hi, everybody, We recently recognized a problem with our Innovaplate SD-2 (a.k.a. MRC 2-Well) plates from Hampton. Our last batch of Innovaplates have an inconsistent well height such that our crystallization robot (a Mosquito) cannot put protein in all the wells. When tested with an older Innovaplate or a hanging drop cover, everything was fine. Is anybody else observing this? I heard that European customers might be getting the same plates from a different source/manufacturing facility. If that is the case, from which company do you buy these plates? Another question is about 96-channel pipetting equipment: For experiments unrelated to crystallography I am in need of getting a 96-channel pipetting machine, such as Rainin's Liquidator. I am also hoping to use this for transferring crystallization screens from blocks into crystallization trays (Our robot sets up only the drops, but does not transfer large volumes of crystallization solutions). Does anybody use the Liquidator for crystallization reagent transfers, and if you do so, would you recommend it? I am open to any brand/model, so all suggestions are welcome. Thanks, Engin -- Engin Özkan Post-doctoral Scholar Laboratory of K. Christopher Garcia Howard Hughes Medical Institute Dept of Molecular and Cellular Physiology 279 Campus Drive, Beckman Center B173 Stanford School of Medicine Stanford, CA 94305 ph: (650)-498-7111
