help! Truncated protein cannot expressed in mammalian cells? - (Mar/09/2009 )
I am trying to study the functional domains in my transcription factor. I cloned the full-length protein with a N-terminal Myc tag in a mammalian expression vector. The protein expressed good verified by western blot with both specific antibody and Myc antibody. When i truncate the protein or take out a domain in the middle of the protein, I cannont dectect the protein fragmemnt with Myc antibody. Does anyone here have any idea of these? The sequencing is correct and in frame. Thanks!
I assume these are denaturing gels? You say that you cannot detect the truncated fusion proteins with the myc antibody. Can you detect them with your protein target antibody still or does that also disappear? If both have disappeared, have you tried producing them by in vitro transcription/translation reactions? Sometimes loss of a protein region or even a few bp changes significantly alters recombinant protein expression, so, perhaps your expression is merely lower than usual. You could try immunopurifying your protein with myc antibodies and protein G beads before loading onto SDS-PAGE to concentrate it. Also, make sure that your recombinant proteins don't have any codon usage problems due to the new fusion sequence.
Dr Teeth on Mar 9 2009, 11:42 AM said:
Dr Teeth, thanks for your reply. My target antibody only detect the c-terminal part of the protein, which means it won't recognize the truncated protein when I delet the c-terminal. For one of the constructs I retained the c-terminal, that antibody couldn't work either. The thing is wether I delet the N or C or even a middle part of the protein, I cannot detect anything. Even when I pull-down with protein G, it didn't show anything. BTW, I fused the proetin fragments with a GFP N-terminal tag as well, it seems the expression of GFP-fused proteins have no problem. I guess I will try the in vitro transcription, but in that way, I cannot do my following experiments.
May be the proteins were unstable due to truncation and degraded. Are you sure the truncated proteins are expressing well??
sharath on Apr 21 2009, 04:23 PM said:
This was my thought as well. You may be getting expression but the mutant is being degraded just as quickly and hence, no detection. Try treating your cells with a proteosome inhibitor and see if you can detect the protein then.
I have done both N C truncation before. Your case could be caused by the degradation of the interested proteins. Be sure to add protease inhibitor after cell lysis on ice and boil the sample immediately. When you add from N terminal, be sure that your tag is in the ORF, no signal peptide that will be deleted after translation. If nothing worked, your N terminal must be very important. I have no other ideas.
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It would be tremendous help to my research if you could share the info about which proteins with which truncations exactly didn't express. I'll acknowledge your help in my paper or cite yours!
please send me an email at hegyihedi AT gmail.com