Hi Firdous, The question, if I am understanding correctly, is to find the topology of the regions of a membrane protein, that is cytoplasmic, membranous or extra-cellular. You can do it by site directed cystine mutagenesis nearby to the the preferred region/domain followed by MAL-PEG analysis. There are several studies using this approach to find the topology of membrane protein. You must put a tag like HA or 3XFLAG nearby to the domain/region. Regarding the difficulties in expressing membrane protein, they are usually toxic for cells in over-expressed condition. So, a careful approach would be growing bacterial culture at lower temperature like 18 degree celcius and also putting lower concentration of IPTG (0.1mM) as the final concentration in culture (if it's a T7 based expression system). Hope this helps.
Best!! Debasish CSIR- Senior Research Fellow (PhD Scholar) C/o: Dr. Akash Ranjan Computational and Functional Genomics Group Centre for DNA Fingerprinting and Diagnostics Hyderabad, INDIA Email(s): dkgh...@cdfd.org.in, dgho...@gmail.com Telephone: 0091-9088334375 (M), 0091-40-24749396 (Lab) Lab URL: http://www.cdfd.org.in/labpages/computational_functional_genomics.html ----- Original Message ----- From: Firdous Tarique <kahkashantari...@gmail.com> To: CCP4BB@JISCMAIL.AC.UK Sent: Mon, 11 Dec 2017 08:03:37 +0530 (IST) Subject: [ccp4bb] domain prediction of a membrane protein Hello everyone I am trying to clone some domains of a transmembrane protein. I have used an online server "Phobius" for the prediction of sequences which is showing it to be a membrane protein with cytoplasmic, non cytoplasmic and transmembrane regions. Attached is the image of that prediction. Regions from 100-200 amino acid residues which I am more concerned about is predicted to be cytoplasmic in nature while other servers have predicted it to be non cytoplasmic in nature. I am having problems in expressing this domain. Sometimes the prediction may go wrong for such a short stretch of amino acid residues. My question is to know about any technique or any classical experiment which can actually tell me the exact location of this domain across the membrane. I mean real experiments not predictions. Regards Firdous Graduate student Delhi University India
