I have isolated and identified more than 40 hypothetical proteins from E. coli by using MALDI-TOF and LC-MS/MS. Hypothetical proteins are cloned, over expressed and two proteins are characterized by binding study and crystal study.
Most of the proteins are not forming crystals. It is very hard to make deletion mutant of all hypothetical genes and make characterization of hypothetical genes. I have isolated and purified most of the hypothetical proteins by using chromatography. I would appreciate if anyone could give suggestions and advice regarding the protein identification by using any functional studies.
To get a general idea about the protein function you can look for homologous proteins which might have been characterized in different organisms.
Once you have a guess what the function might be, you can design an experiment to test that hypothesis.
Thanks for your answer. I have searched my proteins in database and compared the similarity with different organisms. I found that all homologous proteins from different organism has uncharacterized domain and not found any specific domain. Therefore guessing also not possible most of the time. Anyway I ll keep your answer in my mind
In my opinion, it'll be possible to use CD spectroscopy for folding evaluation of your proteins (http://www.mscwu.wur.nl/NR/rdonlyres/256B4909-D151-45EF-BA90-47A81D1F17A0/57402/Kelly2005.pdf; is really handle and helpful). Also some molecular modelling could help, but you should keep going on homology search, maybe you will be luckier
Dear Gnanasekaran
I see you have umpteen HP domains. Could you tell us where they are located? Any specific signature sequences they harbor? Also ensure where they are putatively localized?
I will be glad to help you in this while you may go through these:
http://www.ncbi.nlm.nih.gov/pubmed/?term=Suravajhala+P+AND+hypothetical+proteins
I hope this helps
Prash
I think that is very important a refined bioinformatic study of hypothetical proteins for planning a very detailed set of wet-laboratory experiments
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