After cloning with PET 21 a, and transformation with BL21(DE3) , I confirm the target protein by SDS page. Before SDS technique I detect the protein by Bradford assay method ; ( low protein conc. without IPTG and high protein conc. with IPTG as inducer) .MY question is : why the gene expressed and produce low protein conc. with out IPTG inducer.May be there is a little amount of lactose lead to expression ……… any body can clearfy that please.
How can you tell that your recombinant protein is being produced just by performing a Bradford assay ? What you get is the total amount of E. coli protein present thé crude extract; the only way to tell if the recombinant protein is present is to perform SDS-PAGE analysis and compare the induced vs. the uninduced extract. Western blotting could also be useful for this purpose.
If the recombinant protein is visible on SDS-PAGE without IPTG induction, it implies leaky expression. The expression of gene needs to be tightly regulated and so the promoter needs to be optimized.
Leaky expression can be a common occurrence with lac operon based expression vectors.
https://www.nature.com/scitable/blog/bio2.0/more_on_operons/
The key question to ask yourself is whether this matters? If you only need to express a protein for purification and you are able to do that, then don't worry so much. If you only care because you think you should be a binary expression on a gel then you shouldn't.
People care about leaky expression when the protein itself may inhibit bacterial growth or even result in toxicity. Don't let the tail wag the dog.
Dear Fadhil
which media did you use?
Some basal expression with T7 promoter may happens, expecially in media that not contains glucose (which act as repressor of the T7 promoter) also in absence of lactose.
You can solve this problem (if it is really a problem for you)
1) by change the expression media (eg. use the Enpresso media http://enpresso.de/en/ which is a stach based media where you obtain a slow release of glucose which allows not only a great bacterial growth but also a good and stable promoter repression thanks to the presence of glucose released from the starch.
2) using a plyss strain which thanks to the production of lysozime is more repressed.
if you are interested to know more information abouts enpresso and plyss you can see the following videos:
ProteoCool n°5 (E.coli high cell density media overview
ProteoCool n°22 (Overview on Tools for recombinant protein expression in E.coli
avaialble on my blog
ProteoCools ( http://proteocool.blogspot.com/ )
Ciao
MAnuele
Great thanks for all , dr. Thomas J.Esparaza thanks a lot and I will never let the tail wag the dog and I will go ahead in my project
Dear Fadhil,
Yeah leaky expression, I suppose from your explanation. You have stated lactose; Did you have lactose in your media? In r-expression systems, we collect samples prior to induction (IPTG), called the pre-induction sample, and collect samples, every three hours post induction ( Post Induction 1, 2, and 3). The concentration of protein usually increases, post induction. If this is the case with your expression (never mind the miniscule expression, pre-induction), then, you probably are on the right direction. If you still need higher expressions, then do a HCDC, perform induction and harvest them media (containing protein).
Great thanks Navnit Kumar for you replay...
Do u have any related article ,I need it as a reference.
Dear Fadhil,
Articles, I am not sure of. However, we follow this procedure in our lab. I think one of the old instruction manuals for a pET, which we purchased long back had this. I'll share it to you if I happen to find it.
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