Expression and -N end rule

shifali chatrath via methods%40net.bio.net (by shifalich from rediffmail.com)
Sat May 2 05:52:26 EST 2009


Yeah! I also tgought so, just wanted second opinion out of text books.
You mean to say If Bulkier groups are present next to Met, MAP will not act and Met will stay with the protein sequence?
I checked the expression with coomassie and silver stain. i cudn't do western because the protein was untagged. Also, I checked in medium (Unconc. n conc.), cell lysates ( sup. n pellet both), I could not find band coressponding to my protein or any overexpressed band on the gel.I analysed abt. 6 recombinant colonies and 2 control colonies as negative ctrl.But nothing. Coomassie was blank in medium, no bands at all.

Thanks
Regards
Shifali

On Wed, 29 Apr 2009 17:55:49 +0530  wrote
>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
>> _______________________________________________
>> Methods mailing list
>> Methods from net.bio.net
>> http://www.bio.net/biomail/listinfo/methods
>> 
>

Shifali Chatrath
Graduate Student
Protein science Lab
Dept. of Biological sciences
National University of Singapore
Singapore
+65-65161210
+65-96393449


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