Expression and -N end rule

Alejandro Miguel Martin Dunn via methods%40net.bio.net (by alejandro.martin from cigb.edu.cu)
Wed Apr 29 07:26:57 EST 2009


Shifali,

the N-end rule works at the cytoplasm; it has no influence on the
half-life of proteins secreted into the ER (as should be the case for
your venom protein in Pichia). Also (at least in E. coli) remember that
the amino-terminal methionine is not always removed by MAP; this depends
on the bulkiness of the side chain for the second a.a. Unsurprisingly,
residues which are destabilizing according to the N-end rule often
inhibit removal of the N-terminal Met by MAP when they are in the second
position.

how did you check for expression in yeast? did you also test cell
lysates? Straight SDS-PAGE or Western blotting?

> -----Original Message-----
> From: methods-bounces from oat.bio.indiana.edu 
> [mailto:methods-bounces from oat.bio.indiana.edu] On Behalf Of 
> shifali chatrath
> Sent: Tuesday, April 21, 2009 10:46 PM
> To: methods from magpie.bio.indiana.edu
> Subject: 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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