[ccp4bb] Post-doctoral and PhD position : Structure-function of chemokine receptors
Post-doctoral and PhD position : Structure-function of chemokine receptors Corinne Vivès, Eva Pebay-Peyroula and Franck Fieschi, Institut de Biologie Structurale, UMR5075 CEA-CNRS-Univ. J.Fourier, 41, rue Jules Horowitz, F38027 Grenoble cedex 1, FRANCE In collaboration with Jean-Luc Popot, C.N.R.S./Université Paris-7 UMR 7099, Institut de Biologie Physico-Chimique, 13, rue Pierre-et-Marie-Curie, F-75005 Paris, FRANCE CXCR4 and CCR5 are chemokine receptors and belong to the 7 transmembrane G-Protein coupled receptor (GPCR) family. Besides their physiological functions, they have attracted the scientific community by their role in HIV internalization. So far the identification of HIV inhibitory molecules has been mainly empirical, as their rational design would request the structural characterization of the HIV co-receptors. Structural studies of membrane proteins are highly limited by the bottleneck of their production in quantities compatible with structural studies. In this context we have been involved in a collaboration to develop a method for the high level production of GPCRs in E.coli, taking CXCR4 and CCR5 as a working model. E.coli has proved to be an amazing machinery for protein recombinant expression but is not compatible with the functional expression of eukaryotic membrane proteins. To overcome the problem we address our proteins to inclusion bodies and are now able to produce mg quantities of purified receptors under denaturing conditions. Preliminary tests have already proven the feasibility of the protein refolding. We do provide two positions (one post-doctoral and one PhD) to work jointly on the refolding procedure optimisation using either classical detergent solutions or non-natural surfactants. A biochemical characterisation of the refolded proteins will be required (ligand binding capacity, stability) with the final goal to achieve structural investigations. We have also developed in parallel a functional expression system that does not allow expression yield compatible with structural research but that will enable structure/function studies. The PhD position proposed requires a european student (except French) with a strong biochemistry and/or structural biology knowledge. The 3 year European funding could start before the end of 2008. For the post-doctoral project, we are looking for a graduate student with strong biochemical background, specific skills related to membrane protein biochemistry would be appreciated. A grant from the French National Research Agency will allow an 18 months funding and could start as soon as possible. The research will take place in one of the groups of the Membrane Protein Laboratory headed by Eva Pebay-Peyroula in the IBS (Structural Biology Institute) located within Grenoble scientific polygone ranked in the top european research centers. If you are interested, feel free to contact us to discuss the project to the following address. Do not hesitate to refer to our web sites. [EMAIL PROTECTED] http://www.ibs.fr/content/ibs_eng/home/ http://www.ibs.fr/content/ibs_eng/presentation/lab/lpm/
Re: [ccp4bb] dry shipper on airplaine
Hi Clemens We normally check in the dewar as normal luggage after we have removed all liquids, we have never had any problems travelling from Aarhus to SLS. just remind the students coming along not to mention movies like Twelve monkeys or outbreak when checking it in. best Preben > Dear all, > > I wonder if it would be still/again possible to check in a dry shipper for > a > flight inside Europe. What is your experience? > > Cheers, > Clemens > >
Re: [ccp4bb] Problem with crystallization of Se-Met labeled protein
Consider also the possibility that your SeMet protein differs from the native protein by something other than the sulfur --> selenium. Growing your cells in different conditions may induce different host proteins that could modify your protein. (Often we use rich media for native protein production and minimal media of some flavor for SeMet) We had an example of this, (EJ Drake, Chem Biol, 13, 409) where growing the cells in minimal media (low iron, being the relevant factor) induced a host siderophore biosynthetic pathway that post-translationally added a cofactor to our recombinant protein. We only figured out why we couldn't crystallize the SeMet protein after we finally solved the structure (by a low homology molecular replacement) and saw that the site of cofactor addition was buried against a symmetry related molecule. Adding the 350Da cofactor at this position likely prevented the SeMet crystals from growing. Bottom line, if there is any chance of a post-translational modification, make sure your growth conditions are as similar to native protein expression as possible. A methionine auxotroph may be preferred over metabolic inhibition in this case. Andy On 5/27/08 5:11 PM, "Joe Smith" <[EMAIL PROTECTED]> wrote: > Dear all, > Sorry for an off-topic query. > I have been unable to crystallize a Se-met containing protein (8 Met > in 206 amino acids) in the native crystallization condition ( 0.1 M > Tris pH 8.5, 1.2 M K-Na-tartrate; Theoretical pI of protein is 8.4). > As expected, solubility of Se-Met containing protein is little less > than the wild type. Other than seeding, i don't know what else I > should try for obtaining a Se-met crystal for phasing. Can I mutate > some of the exposed Met (based on secondary structure prediction and > homologous structure) to Ala as I feel I don't really need 8 Se for > phasing 208 aa long polypeptide. I want to know what generally one > should do when Se-Met containing proteins fail to crystallize. > Thanks in advance. > Joe > PS: Since, protein contains 3 Cys residues.. I am also planning to try > my luck with heavy atom compounds containing Hg. -- Andrew M. Gulick, Ph.D. --- (716) 898-8619 Hauptman-Woodward Institute 700 Ellicott St Buffalo, NY 14203 --- Senior Research Scientist Hauptman-Woodward Institute Dept. of Structural Biology, SUNY at Buffalo http://www.hwi.buffalo.edu/Faculty/Gulick/Gulick.html http://labs.hwi.buffalo.edu/gulick
[ccp4bb] insect expression system
Hi, I searched my mail box for possible answers you have given but couldn't find any. The question is about selecting insect expression systems for mammalian or viral glycosylated proteins. There is confirmed expression of the target proteins in mammalian cells. They are secreted into the media. 1) Baculovirus system 2) Drosophila system Which system would you recommend for high expression and convenience? Has anyone ever compared those systems side by side? Any suggestions or references are greatly appreciated. Yongfu Li
Re: [ccp4bb] insect expression system
Hi, with S2 you can get away without figuring out if the virus works, then again you probably need a stable cell line... (so which ever works...?) (OBS! cant do SeMet labelling with S2!! or if someone can please tell me) -Tommi Quoting Yong-Fu Li <[EMAIL PROTECTED]>: > Hi, > > I searched my mail box for possible answers you have given but couldn't > find > any. The question is about selecting insect expression systems for > mammalian > or viral glycosylated proteins. There is confirmed expression of the > target > proteins in mammalian cells. They are secreted into the media. > > 1) Baculovirus system > 2) Drosophila system > > Which system would you recommend for high expression and convenience? > Has anyone ever compared those systems side by side? > > Any suggestions or references are greatly appreciated. > > Yongfu Li > -- Tommi Kajander, Ph.D. Macromolecular X-ray Crystallography Research Program in Structural Biology and Biophysics Institute of Biotechnology P.O. Box 65 (Street address: Viikinkaari 1, 4th floor) University of Helsinki FIN-00014 Helsinki, Finland Tel. +358-9-191 58903 Fax +358-9-191 59940
Re: [ccp4bb] insect expression system
I searched my mail box for possible answers you have given but couldn't find any. The question is about selecting insect expression systems for mammalian or viral glycosylated proteins. There is confirmed expression of the target proteins in mammalian cells. They are secreted into the media. 1) Baculovirus system 2) Drosophila system Which system would you recommend for high expression and convenience? For most proteins, the level of expression is far greater with bacuovirus. Dima
[ccp4bb] BEAM TIME AVAILABLE FOR MACROMOLECULAR CRYSTALLOGRAPHY
BEAM TIME AVAILABLE FOR MACROMOLECULAR CRYSTALLOGRAPHY AT THE TWO ID BEAMLINES OF NE-CAT AT APS. Beamline 24ID-C: Variable energy between 6-18keV, ADSC Q315 Detector, MAD Capable. Beamline 24ID-E: Equipped with MD2 microdiffractometer, delivering beam as small as 5 microns. Single energy (12662eV), ADSC Q315 Detector. Contact [EMAIL PROTECTED] for further details. See http://necat.chem.cornell.edu for information on our beamlines and a calendar of available days.
Re: [ccp4bb] insect expression system
Hi, It is called Bacmam. Please see the following reference, it is not the land mark reference, if you dig pubmed you should be able to find the first description of the system. BacMam recombinant baculovirus in transporter expression: a study of BCRP and OATP1B1. Protein Expr Purif. 2006 Jun;47(2):591-8. Epub 2006 Jan 30. Hope this is what you were looking for Cheers Pius Stephen Padayatti On Wed, May 28, 2008 at 10:40 AM, Yong-Fu Li <[EMAIL PROTECTED]> wrote: > Hi, > > I searched my mail box for possible answers you have given but couldn't find > any. The question is about selecting insect expression systems for mammalian > or viral glycosylated proteins. There is confirmed expression of the target > proteins in mammalian cells. They are secreted into the media. > > 1) Baculovirus system > 2) Drosophila system > > Which system would you recommend for high expression and convenience? > Has anyone ever compared those systems side by side? > > Any suggestions or references are greatly appreciated. > > Yongfu Li -- Pius S Padayatti Department of Biochemistry, Structural Biology Division, School of Medicine, RT-500 Case Western Reserve University, Cleveland, Ohio-44106, 216-368-6833
Re: [ccp4bb] insect expression system
Dear Yong-fu, Why would you worry about insect expression systems if you already can secrete your constructs in mammalian cells? For such proteins, transient expression in HEK cells for example gives higher yields than baculo, is faster, cheaper, you can nicely control glycosylation, easily do Se-Met labelling and so on. Here are some references (PMID): 17355862, 17001101, 16823037, 11788735, 16082028. Radu -- A. Radu Aricescu, PhD University of Oxford Wellcome Trust Centre for Human Genetics Division of Structural Biology Roosevelt Drive, Oxford OX3 7BN United Kingdom Phone: +44-1865-287551 Fax: +44-1865-287547 Original message >Date: Wed, 28 May 2008 10:40:16 -0400 >From: Yong-Fu Li <[EMAIL PROTECTED]> >Subject: [ccp4bb] insect expression system >To: CCP4BB@JISCMAIL.AC.UK > > Hi, > > I searched my mail box for possible answers you have > given but couldn't find any. The question is about > selecting insect expression systems for mammalian or > viral glycosylated proteins. There is confirmed > expression of the target proteins in mammalian > cells. They are secreted into the media. > > 1) Baculovirus system > 2) Drosophila system > > Which system would you recommend for high expression > and convenience? > Has anyone ever compared those systems side by side? > > Any suggestions or references are greatly > appreciated. > > Yongfu Li
Re: [ccp4bb] insect expression system
Hi Yongfu, I couldn't agree more with Radu. We had great success in expressing mg amounts of a secreted protein in HEK293 cells (with Radu's help :-) . The same protein was initially expressed in Sf9 cells but with much lower yields. Furthermore, we could very easily generate a stable HEK293 cell line expressing the same protein (at similar levels with transient transfections) with the Flp-In system in just a couple of weeks. We also have a LIC vector which is compatible with the Flp-In system. Hope this helps, Vangelis -- Vangelis Christodoulou Analist C Anastassis (Tassos) Perrakis lab The Netherlands Cancer Institute (B8.038) Plesmanlaan 121, 1066 CX, Amsterdam The Netherlands tel: +31 (0)20 512 1963 Website: xtal.nki.nl/Tassos_group/ On May 28, 2008, at 7:36 PM, A. Radu Aricescu wrote: Dear Yong-fu, Why would you worry about insect expression systems if you already can secrete your constructs in mammalian cells? For such proteins, transient expression in HEK cells for example gives higher yields than baculo, is faster, cheaper, you can nicely control glycosylation, easily do Se-Met labelling and so on. Here are some references (PMID): 17355862, 17001101, 16823037, 11788735, 16082028. Radu -- A. Radu Aricescu, PhD University of Oxford Wellcome Trust Centre for Human Genetics Division of Structural Biology Roosevelt Drive, Oxford OX3 7BN United Kingdom Phone: +44-1865-287551 Fax: +44-1865-287547 Original message Date: Wed, 28 May 2008 10:40:16 -0400 From: Yong-Fu Li <[EMAIL PROTECTED]> Subject: [ccp4bb] insect expression system To: CCP4BB@JISCMAIL.AC.UK Hi, I searched my mail box for possible answers you have given but couldn't find any. The question is about selecting insect expression systems for mammalian or viral glycosylated proteins. There is confirmed expression of the target proteins in mammalian cells. They are secreted into the media. 1) Baculovirus system 2) Drosophila system Which system would you recommend for high expression and convenience? Has anyone ever compared those systems side by side? Any suggestions or references are greatly appreciated. Yongfu Li
Re: [ccp4bb] odd waters
None except 8 Se. So either - a) L,K edge far below, or b) K-edge above 12.7keV. Cl fits a) and refines to B-factors close to the sourrounding protein atoms. Thx, br _ From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of Abhinav Kumar Sent: Tuesday, May 27, 2008 8:44 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] odd waters Check the anomalous difference map on these sites. Thanks Abhinav Abhinav Kumar JCSG @ SSRL, MS 99 2575 Sand Hill Road Menlo Park, CA 945025-7015 Phone: 650 926 2992 Fax: 650 926 3292
[ccp4bb] Postdoctoral opening at UT Southwestern
A postdoctoral position combining structural/biophysical techniques and protein engineering to discover the molecular mechanisms that control microtubule assembly is available immediately in Luke Rice’s lab in the Department of Biochemistry at UT Southwestern Medical Center. The lab is currently focused on enabling structural and mechanistic studies of alpha/beta-tubulin by harnessing the established (but largely untapped) potential of S. cerevisiae as a vehicle for recombinant expression of wild-type or mutant alpha/beta-tubulin. The ideal candidate will show strong motivation and will have recently earned a Ph.D. with experience in structural biology and/or molecular biology and protein expression. Applicants should email a CV along with contact information for three references and a brief description of their previous research. -- Luke M. Rice Assistant Professor Department of Biochemistry, ND10.300 UT Southwestern Medical Center 5323 Harry Hines Blvd. Dallas, TX 75390-8816 phone: (214) 645-5931 email: [EMAIL PROTECTED]
[ccp4bb]
Hi, Can anyone direct me to a program that calculates a protein cavity's dimensions (average) and not just its volume? Thanks in advance, Xie
