Overexpression of a protien (fwd)

nico at lmb1.rug.ac.be nico at lmb1.rug.ac.be
Thu Nov 9 09:18:46 EST 1995


In article <Pine.SOL.3.91.951025101344.12308B-100000 at welchlink.welch.jhu.edu> tlu at WELCHLINK.WELCH.JHU.EDU (Natalie Lu) writes:
>From: tlu at WELCHLINK.WELCH.JHU.EDU (Natalie Lu)
>Subject: Overexpression of a protien (fwd)
>Date: 25 Oct 1995 13:28:24 -0700


>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.

A problem with the T7 RNA based expression system is the level of 
repression: even a small amount of leaky expression of the T7 RNA polymerase 
gene can result in an onset of induction. It then depends on the toxicity of 
the product and/or the burden of the system on the cells what your outcome 
will be. Sometimes you get good inductions, sometimes less good, sometimes you 
see nothing, although the plasmids can be forced to remain in the cell. What 
you actually do is select for expression-down mutants.
You can lower basal-level expression by co-expression of a lysozyme that 
turned out to be an inhibitor of the T7 RNA polymerase (pLysS). Since this 
lysozyme is not well appreciated by the host, this also increases the burden on
the cell.
We have described a well-repressed, plasmid based induction system for the T7 
RNA polymerase in:

Mertens, Remaut and Fiers (1995): Tight transcriptional control mechanism 
ensures stable high-level expression from T7 promoter-based expression 
plasmids. BIO/TECHNOLOGY 13:175-179

In the paper we also present proof for the statement I made above.
You can request the plasmids or give your comments on the paper by E-mail:
nico at lmb1.rug.ac.be
good luck,

Nico Mertens
Lab. Molecular Biology
Ledeganckstraat 35
B-9000 Gent
Belgium






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