The T7 expression system inducible by IPTG is very leaky. The problem could
be that your protein is a bit toxic to the cells so the expression level is
kept low. One of the solutions is use of bacterial strains containing pLysS
or pLysE plasmid (e.g. BL21 (DE3) pLysS or E). Both plasmids carry gene
encoding lysozyme, which inhibits strongly T7 polymerase. Difference between
S and E is in the level of expression of lysozyme. In this system there
should be almost no basal expression of your gene (but it is just a theory -
if your protein is really toxic better use another system).
Another problem comming with such bacterial expression system is varying
expression level. When you want to choose best expressing clone you shoud
screen at least 10 to 20 colonies. Once I screened 40 colonies and only one
was really good expressing.
Hope this helps
Vladimir Rotrekl
Dept. of Biochemistry
Faculty of Science
Masaryk University
Kotlarska 2
61137 Brno
Czech Republic
Tel.: +425 41129374
Fax: +425 41129372
Kelly Luther wrote in message <36F865B8.F8090F27 at cc.umanitoba.ca>...
>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>******************************************************************
