IUBio

protein expression in PLysS

Kelly Luther umluther at cc.umanitoba.ca
Tue Mar 23 23:10:32 EST 1999


kandasamy ravi wrote:
> 
> Sir,
> 
>    I have cloned my gene in pET15b transformed in to DH5 and isolated DNA and
> retransformed in to pLysS BL21(DE3).checked the sequences(it is in right frame)
> But i don't get overexpression just little increase in expression.and the uninduced
> one is expressing as good as induced.I tried different IPTG concentration but no
> use and even the story is same in C43/C41 cell lines.
>     what do i do to get over expression?and why there is a leaky expression in
> uninduced control.
> somebody help me please...
> All suggestion welcome
> 
> Thank you
> K.Ravi

The first question is how do you know you are getting expression from
the non-induced control?  Am I to presume you have western blots with
similar bands?  If not, you may not be getting any expression.

First I would try growing at a lower temperature, say 30 degrees.  Other
than that, the obvious changes such as altering the amount of IPTG from
0.4 mM to 1mM or more, and altering induction time between an O.D.600 of
0.4 to 0.8.  You may also be getting degradation.  Are you doing a time
course to look for your protein?  You may have to harvest 2 hours post
induction as opposed to 4 or more for example.

You can also check to insure that there are no rare codons at the
beginning of the gene.  Multiple rare codons can be particularly harmful
at the start of your gene.

If pET15 is like my vector, the HIS tag can be placed at either end of
the gene.  Try putting it at the N-terminus, which can significantly
increase over-expression (don't ask me why!).

I believe you can also have problems with improper processing of
N-terminal fMet in E. coli, but I would have to look that one up.  If
this is a potential problem with your protein you may want to look into
this.  

Have you done a growth curve?  If so what did it look like after you
induced?  If the cells start dying right away then you may have to try
other strains that grow better when induced.

-- 
******************************************************************
Kelvin Luther
Department of Chemistry         "Illegitimati non carborundum"
University of Manitoba            General Joseph W. Stilwell
Winnipeg, MB, Canada
mailto:umluther at cc.umanitoba.ca
******************************************************************



More information about the Methods mailing list

Send comments to us at biosci-help [At] net.bio.net