Expression and -N end rule

Suresh Kumar via methods%40net.bio.net (by kumar from lifesensors.com)
Wed Apr 22 14:40:45 EST 2009


Hi Shifali,

Your protein is stable in the snake venom, because the venom itself
might not have the 26S proteasomal activity. I think venom proteins are
secreted using secretion granules which shield them from the cellular
26S proteasomal activity.

Regarding your troubles with expression, you could try these options.

1- Codon optimization. For a small protein such as yours the entire gene
could be synthetically made with codon optimization for
E.coli/Yeast/Insect with affordable price (at least here in the US).

2-SUMO expression system- Look into the SUMO expression system. 

http://lifesensors.com/www/sumo-expression-systems-c-55.html

In this system, SUMO (Small Ubiquitin-like Modifier) tag is added to the
N-terminus of your protein. This will prevent N-end rule degradation if
there is any. Once the protein is expressed you can purify it using
standard Ni-Affinity Purification (There is a His tag N-terminus to
SUMO). SUMO tag is then cleaved with a SUMO specific protease which will
leave the original N-terminus of your protein intact (Arginine in your
case). 

This system offers vectors for E.coli, Yeast and Insect Cells.

3- Once codon optimized, you can try several standard conditions to
optimize the expression.

-Medium
-IPTG (Low to High)
-Temperature (18oC to 37oC)
-Time
-Lysis buffers
-Protease/Proteasome inhibitors

Hope this helps and best of luck!

Suresh

Disclaimer: I do not work for Life Sensors Inc., but my company has
close ties with them and I have used the SUMO expression system
successfully in E.coli. 

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-----Original Message-----
From: methods-bounces from oat.bio.indiana.edu
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Sent: Wednesday, April 22, 2009 1:05 PM
To: methods from magpie.bio.indiana.edu
Subject: Methods Digest, Vol 47, Issue 16

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Today's Topics:

   1. phenol in alcohol precip (Ed Siefker)
   2. Blue/white with pET15b, Rosetta2(DE3)? (oliver.starks from gmail.com)
   3. Genomic DNA isolation from mammalian cells (Dwayne Taliaferro)
   4. Re: Blue/white with pET15b, Rosetta2(DE3)? (DK)
   5. Re: phenol in alcohol precip (WS)
   6. Re: Genomic DNA isolation from mammalian cells (DK)
   7. Expression and -N end rule (shifali  chatrath)
   8. Re: Expression and -N end rule (WS)
   9. Genomic DNA isolation from mammalian cells (clarification)
      (Dwayne Taliaferro)


----------------------------------------------------------------------

Message: 1
Date: Tue, 21 Apr 2009 08:38:04 -0500
From: Ed Siefker <ebs15242 from creighton.edu>
Subject: phenol in alcohol precip
To: methods from magpie.bio.indiana.edu
Message-ID: <49EDCC3C.2000906 from creighton.edu>
Content-Type: text/plain; charset=ISO-8859-1; format=flowed

Hi.  I was a little hasty in doing some phenol/chloroform
preps of genomic DNA and apparently didn't remove all
the phenol before adding ethanol to precipitate.  How can
I recover from this?

I considered extracting with chloroform again, but I don't
know if the phases will still separate with ethanol in there.
Also, the DNA isn't in solution, so I'm guessing it would
collect at the interface.  If there were more DNA there,
I'd just fish it out with a pipette tip, but it's a small prep.

Any thoughts?  Thanks a bunch
-Ed



------------------------------

Message: 2
Date: Tue, 21 Apr 2009 07:26:50 -0700 (PDT)
From: oliver.starks from gmail.com
Subject: Blue/white with pET15b, Rosetta2(DE3)?
To: methods from net.bio.net
Message-ID:
	
<c86f0875-c90c-47ea-bf71-5d0b8b824659 from s31g2000vbp.googlegroups.com>
Content-Type: text/plain; charset=ISO-8859-1

I've got my gene inserted in pET15b, and I transformed Rosetta2(DE3).
Colonies appeared in appropriate numbers on an Amp/Cam plate,
indicating the cells must have the vector.  Without thinking it out I
re-streaked a couple colonies onto another plate with IPTG/Xgal to re-
test insert presence (I'm fairly confident in my original ligation but
it's always possible that the cells from which I prepped my plasmid
were cotransformed with empty pET15b as well).  These produced blue
coloration.  But after thinking about it more, it seems like either
blue should never be produced (regardless of insert presence/absence)
or should always be produced (regardless of insert presence/absence) -
because the insert is not disrupting a lacZ gene like it is in
pBluescript or something of that nature.

So...how's the blue being produced, and is it even evidence of my
insert not being present?

I'll be testing these with new preps and PCR or digest today or
tomorrow anyway but thanks in advance for your help.


------------------------------

Message: 3
Date: Tue, 21 Apr 2009 13:20:43 -0400
From: Dwayne Taliaferro <taliaferroD from mail.nih.gov>
Subject: Genomic DNA isolation from mammalian cells
To: <methods from magpie.bio.indiana.edu>
Message-ID: <C61378AB.306E%taliaferroD from mail.nih.gov>
Content-Type: text/plain;	charset="US-ASCII"

Does anyone have a quick and reliable protocol for isolating gDNA from
mammalian cells?  I  would be interested.



------------------------------

Message: 4
Date: Tue, 21 Apr 2009 22:59:52 GMT
From: dk from no.email.thankstospam.net (DK)
Subject: Re: Blue/white with pET15b, Rosetta2(DE3)?
To: methods from net.bio.net
Message-ID: <BdsHl.83185$9t6.49292 from newsfe10.iad>

In article
<c86f0875-c90c-47ea-bf71-5d0b8b824659 from s31g2000vbp.googlegroups.com>,
oliver.starks from gmail.com wrote:
>I've got my gene inserted in pET15b, and I transformed Rosetta2(DE3).
>Colonies appeared in appropriate numbers on an Amp/Cam plate,
>indicating the cells must have the vector.  Without thinking it out I
>re-streaked a couple colonies onto another plate with IPTG/Xgal to re-
>test insert presence (I'm fairly confident in my original ligation but
>it's always possible that the cells from which I prepped my plasmid
>were cotransformed with empty pET15b as well).  These produced blue
>coloration.  But after thinking about it more, it seems like either
>blue should never be produced (regardless of insert presence/absence)
>or should always be produced (regardless of insert presence/absence) -
>because the insert is not disrupting a lacZ gene like it is in
>pBluescript or something of that nature.
>
>So...how's the blue being produced, and is it even evidence of my
>insert not being present?

Rosetta(DE3) is simply BL21(DE3)  with pRARE2 plasmid. 
The strain has WT lacZ, so naturally it makes blue colonies. 
It has nothing to do with your plasmid or it having an insert. 

DK


------------------------------

Message: 5
Date: Tue, 21 Apr 2009 14:09:10 -0700 (PDT)
From: WS <novalidaddress from nurfuerspam.de>
Subject: Re: phenol in alcohol precip
To: methods from net.bio.net
Message-ID:
	
<a4cb9874-cd47-477e-b2f5-f693c46b21d7 from h28g2000yqd.googlegroups.com>
Content-Type: text/plain; charset=ISO-8859-1

Dear Ed,

don't worry. Just rinse your pellet a few times with EtOH (and spin
down shortly to make sure you don't loose the pellet).. If you plan
downstream processing with enzymes, make sure all phenol has been
removed. You might dare to check thesmell; your nose should turn out
to be a very sensitive GC system with a neuronal detector attached to.
Chloroform might not be that good choice, even that DNA doesn't
dissolve in it, (regardless if there is EtOH and or phenol present),
as it does not dissolve phenol as well as ethanol does. You might
remember: the DNA (and other water soluble stuff) is in the phenol
phase, lipids are in the chloroform phase and proteins are inbetween.

Have fun!

Wo


------------------------------

Message: 6
Date: Tue, 21 Apr 2009 23:22:51 GMT
From: dk from no.email.thankstospam.net (DK)
Subject: Re: Genomic DNA isolation from mammalian cells
To: methods from net.bio.net
Message-ID: <8zsHl.69302$ua7.56187 from newsfe17.iad>

In article <mailman.979.1240346807.13724.methods from net.bio.net>, Dwayne
Taliaferro <taliaferroD from mail.nih.gov> wrote:
>Does anyone have a quick and reliable protocol for isolating gDNA from
>mammalian cells?  I  would be interested.

The purpose of gDNA? For "high" yeild and reasonable purity,
here is what I used on many different cells (dicty, insect, 
mammalian lines):

Pellet cells from 10 ml culture, resuspend in 1% Triton X100, 
spin, keep pellet, lyse nuclei with 400 ul 1% SDS, 20 mM EDTA,
add 600 ul chloroform, vortex 15", spin hard, take upper phase, repeat 
extraction 2X. Add RNAse to 0.1 mg/ml, let sit for 5', mix 3 volumes 
of Qiagen buffer GQ or 5 volumes of Qiagen buffer PB with one 
volume of chloroform-extracted material, load onto regular Qiagen
miniprep column, wash 2X with PB, wash 2X with PE, elute with 
100 ul of 1-10 mM Tris, pH 8.0-8.5 (or TE) heated to ~ 70C. 

DK


------------------------------

Message: 7
Date: 22 Apr 2009 02:45:33 -0000
From: "shifali  chatrath" <shifalich from rediffmail.com>
Subject: Expression and -N end rule
To: <methods from magpie.bio.indiana.edu>
Message-ID: <20090422024533.56211.qmail from f4mail204.rediffmail.com>
Content-Type: text/plain; charset="ISO-8859-1"

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

------------------------------

Message: 8
Date: Wed, 22 Apr 2009 01:13:52 -0700 (PDT)
From: WS <novalidaddress from nurfuerspam.de>
Subject: Re: Expression and -N end rule
To: methods from net.bio.net
Message-ID:
	
<5db5bd9e-8539-425c-8f06-5da81a395164 from m19g2000yqk.googlegroups.com>
Content-Type: text/plain; charset=ISO-8859-1

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


------------------------------

Message: 9
Date: Wed, 22 Apr 2009 11:03:41 -0400
From: Dwayne Taliaferro <taliaferroD from mail.nih.gov>
Subject: Genomic DNA isolation from mammalian cells (clarification)
To: <methods from magpie.bio.indiana.edu>
Message-ID: <C614AA0D.30A6%taliaferroD from mail.nih.gov>
Content-Type: text/plain;	charset="US-ASCII"

I am looking for a protocol that will allow me to purify genomic DNA
from
mammalian cells with the purpose of using it for Southern blotting and
PCR.

DT



------------------------------

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