Overexpression of a protien (fwd)

Enter Your Name Here user at host.uci.edu
Thu Oct 26 01:43:41 EST 1995


In article
<Pine.SOL.3.91.951025101344.12308B-100000 at welchlink.welch.jhu.edu>,
tlu at WELCHLINK.WELCH.JHU.EDU (Natalie Lu) wrote:

> ---------- Forwarded message ----------
> Date: Tue, 17 Oct 1995 17:23:34 -0400 (EDT)
> From: Natalie Lu <tlu at welchlink.welch.jhu.edu>
> To: methods at net.bio.net
> Subject: Overexpression of a protien (fwd)
> 
> 
> I have problems to overexpress my protein. The gene (2kb) was 
> successfully cloned into pET21-a(+) vector. The cloned gene was excised 
> by the same two enzymes (Nde I and XhoI). I saw  the insert on the gel 
> having the correct weight. The DNA sequence of the cloned plasmid is 
> perfect. The insert was cloned into the NdeI site of the vector. Then,the 
> the plasmid was tramsformed into BL21(DE3) cells. The clone was picked to 
> test the overexpression of the protein. I didnot see any overexpressed 
> protien at all. The conditions for grow the cells is as follows: LB 
> medium containing 100ug/ml ampicillin, when O. D. reaches 0.4-0.6, 100mM 
> IPTG was added to a final concentration of 1 mM. Aliquots (500ul) culture 
> was removed every one hour upto 5 hours. The left culture was allowed to 
> grow overnight.No major protien band was found on the SDS gel compared to 
> the uninduced control.  If anyone has any suggestions about this, I will be 
> appreciated. Thanks.

First double check the sequence of the 5' end of your gene where you
subcloned it into pET21. I have seen some weird things happen during
ligations. You should verify that your gene is in frame with the start
codon. I have tried using a 6-his tag vector where the tag was on the 5'
end and the system would just not express my protein even though I could
get a control to work. Try another protein if you have a cDNA for one in
the lab. It seems that bacteria can be sensitive to the sequence of the
fusion proteins so you may have to play around a bit. The two systems that
seem to work well (at least for expression) are the GST vectors from
Pharmicia and the MBP system from NEB someone in your department should
have one of these vectors. Try letting your cells grow up to an OD of 1.0
-1.4 and then induce with IPTG and the final concentration of IPTG can
range from 1mM - 0.05mM depending upon solubility problems that WILL
arrise once you get expression. BL21 are good cells so stick with them.
Also you may want to switch to carbenicillin instead of ampicillin since
carbenicillin is less labile it will ensure antibiotic selection.  Finally
you may not see a huge band on the gel so take a larger aliquot of cells,
lyse them and pass the supernatent over the resin (Ni in your case), elute
in a small volume and run that on a gel that should enhance the amount of
the expressed protein.


good luck, 

Mike Yarski
UC Irvine
mayarski at uci.edu



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