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Expression and -N end rule

Expression and -N end rule - Protocols and Methods Forum

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Old 04-22-2009, 02:45 AM
shifali chatrath
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Default Expression and -N end rule



Dear all!
I had mentioned my problem earlier also in the forum but nobody replied. I am sure you great minds should have a solution to my problem. Please help me if you can!

I want to express a snake venom protein,~ 10kDa, 10 cys residues and 5 disulphide bonds.Mature protein has Arg as first residue after signal sequnece is clipped off.
First of all, i tried yeast expression system.
I expressed my protein in pPICZ alpha, under alpha factor signal sequence followed by Kex2 and Ste13 signal sequences, such that the mature protein will have native -N terminus with Arg as first residue, as of my protein. BUt, I could not see any protein expression even under different medium and proper aeration and whatever possible troubleshooting one could do.
Recently, i read -N end rule which states that Arg is destabilizing residues and directs the protein for degradation to 26S proteasome.
I checked with the company, they say, It hold true for every expression system.
But, my question is, if that is the case, how the protein is stable in snake's venom. I have found the protein in the crude venom of the snake.
Also, the protein is expressed under alpha factor signal sequence which is supposed to be secreted into the medium. Therefore,once the protein is out into medium, it is out of cell now, Kex2 and Ste13 cleavages should be happening outside the cell, therefore, there should be no proteasomal degradation. Am I right?

After clipping off signal sequence, protein will be exported out, still will there be any proteasomal targetting of proteins with destabilizing residue at -N terminus?

I read in -N end rule that, protein's life depends upon penultimate residue to Met. We know, for a protein to be synthesised, we need to have a start codon,i.e. Met. In -N end rule they say, Met will be removed be Metaminopeptidase(MAP), so if we express a protein we should not be counting Met residue in the expected MW? Because, not all mature protein sequences start from Met.


Then,I tried expressing my protein using E. coli epression system, cDNA cloned in pET19b in Rosetta gami cells and RIL cells.I couldn't see any expression. Cells were growing normaly.(I hope, the protein is not inhibiting transcription and translation). Met and Gly had to be added at -N terminus to maintain the reading frame.
Protein is 10Kda and was cloned without tag sequence. I mean expected protein should be 10kDa.I confirmed the sequences before cloning and even plasmid isolated from ex-pression hosts. DNA is there but no protein.

Please answer any, if not all, of the above question. i am struggling with all this for alst 8 months. Your suggestions and help will be highly appreciable.

Thanks in advance.

Shifali



Shifali Chatrath
Graduate Student
Protein science Lab
Dept. of Biological sciences
National University of Singapore
Singapore
+65-65161210
+65-96393449
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  #2  
Old 04-22-2009, 08:13 AM
WS
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Default Expression and -N end rule

Hi Shifali, maybe your protein is toxic? you could change codon usage
and/or try higher eucaryotic cells.

To narrow down the effect of signal sequences etc.: Have you
considered putting your protein (i.e its cDNA) and/or part of it in
front of a reporter gene like GFP or insert just anything known in
order to disrupt a possile action?

Best regards,

Wo
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