yes limited proteolysis is the best choice for the domain determination of unknown protein from our lab some people did this. after doing limited proteolysis just sequence digested part N terminally or the C terminally and find out the region from where its getting digested. side by side u can do fold prediction using phyre or u can use fold index to know the compact part of the protein.
go through J Biol Chem. <http://www.ncbi.nlm.nih.gov/pubmed/18974091> 2008 Dec 26;283(52):36532-41. Epub 2008 Oct 30. Characterization of Rv3868, an essential hypothetical protein of the ESX-1 secretion system in Mycobacterium tuberculosis. Luthra A<http://www.ncbi.nlm.nih.gov/pubmed?term=%22Luthra%20A%22%5BAuthor%5D> , Mahmood A<http://www.ncbi.nlm.nih.gov/pubmed?term=%22Mahmood%20A%22%5BAuthor%5D> , Arora A<http://www.ncbi.nlm.nih.gov/pubmed?term=%22Arora%20A%22%5BAuthor%5D> , Ramachandran R<http://www.ncbi.nlm.nih.gov/pubmed?term=%22Ramachandran%20R%22%5BAuthor%5D> . did similar work . On Fri, Mar 18, 2011 at 4:19 AM, Xianhui Wu <wuxian...@gmail.com> wrote: > Dear all, > > Thank you very much for all of information about domain determination! I > think the limited proteolysis is a good choice for the domain determination > as we have no information about a protein. > > However, if we do have a domain information by the bioinformatics, how > can we truncate the domain? Are we need to keep three or five more amino > acids in two ends? Thanks. > > Best regards, > Xianhui > > > On Tue, Mar 8, 2011 at 2:22 AM, Mark J van Raaij > <mjvanra...@cnb.csic.es>wrote: > >> for an experimental way to determine soluble domains see the following >> paper: >> ESPRIT: an automated, library-based method for mapping and soluble >> expression of protein domains from challenging targets. >> Yumerefendi H, Tarendeau F, Mas PJ, Hart DJ. >> J Struct Biol. 2010 Oct;172(1):66-74. Epub 2010 Mar 4. Review. >> PMID: 20206698 [PubMed - indexed for MEDLINE] >> >> >> >> Mark J van Raaij >> Laboratorio M-4 >> Dpto de Estructura de Macromoleculas >> Centro Nacional de Biotecnologia - CSIC >> c/Darwin 3, Campus Cantoblanco >> E-28049 Madrid, Spain >> tel. (+34) 91 585 4616 >> >> http://www.cnb.csic.es/content/research/macromolecular/mvraaij/index.php?l=1 >> >> >> >> On 7 Mar 2011, at 19:18, gauri misra wrote: >> >> > Hi, >> > To start with it would be great if you look in to the secondary >> structure prediction of the sequence using any of the standard servers like >> PSIPRED, JPRED etc. Many more available at expasy site >> http://ca.expasy.org/tools/. >> > Whatever construct you finally choose to make just remember the standard >> rule that we generally follow is to avoid deleting the alpha helices and >> beta sheets. You can design your initial primers so as to obtain the >> complete amplification of these secondary structures from any part within >> the protein. >> > You can even use the various modules of the following online available >> server >> > http://scratch.proteomics.ics.uci.edu/ >> > to have an idea of the intrinsically disordered regions in the protein, >> transmemebrane regions and disulfide bonds that would certainly help you in >> initiating in the right direction. >> > >> > Best wishes >> > Gauri >> > >> > On Mon, Mar 7, 2011 at 4:10 AM, Xianhui Wu <wuxian...@gmail.com> wrote: >> > Dear all, >> > >> > Before we try to study the crystal structure of an unknown protein, we >> need to determine the sequence that can fold into a compact and stable 3D >> domain. What kinds of methods can we choose? >> > >> > -- >> > Best regards, >> > XH Wu >> > >> > > > > -- > Best regards, > Xianhui > -- Vandana Kukshal Postdoc Fellow structure biology group international Center For Genetic Engineering and Biotechnolgy Aruna Asaf Ali Marg 110 067 New Delhi INDIA
