In a case where we carried out a genetic Screening of genes that involved in the stress response in E. coli. From this screen we identified a gene that encodes a 50 kDa flexible protein (Protein X), which does not have any primary sequence-based similarities with known proteins. So may i know what we need to do next to understand the molecular mechanism of protein X? and How can we deal with this?
It is a very challenging problem. I think I would start by trying to identify other proteins with which Protein X interacts, and I would check to see if it is in an operon.
For the interaction study, you could prepare an antibody against X and use it to co-immunoprecipitate partners of X, then identify them by mass spec sequencing.
Mahesh: Many important proteins (even with their sequences known) in the biological system do not have their function(s) known, and one of which is the amyloid beta in Alzheimer disease. Your task is to assay known functions for Protein X and expect negative results to eliminate certain possibilities. It is a challenge that gives you fun as well as frustrations. Good luck.
It is a very challenging problem. I think I would start by trying to identify other proteins with which Protein X interacts, and I would check to see if it is in an operon.
For the interaction study, you could prepare an antibody against X and use it to co-immunoprecipitate partners of X, then identify them by mass spec sequencing.
Hi Mahesh! You have many options. First, it s not clear what is the source of the protein. If it has no homology to E. coli genome, then it can viral origin. So, look for a virus. Second, if the protein is encoded by E. coli, then you could try knocking it out and check for the outcome e.g. changes in generation time, cell volume, colony size, colour, colony shape etc. Third, overexpression of protein X. Fourth, you can analyze transcriptional networks in knock-outs vs. wild-types vs. overexpression. Fifth, as Adam suggested - make antibodies to protein X and search for its protein partners (or lipids or nucleic acids or sugars).
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