I had same problem with pet42a
Firstly, I was thinking that operon didn't get turned on and this is
why my protein is not getting expressed. Then, I seperated the soluble
and insoluble fratcion by using B-PER regaent from PIERCE company.
Then, I run PAGE-gel and I observed my protein in the insoluble
fraction. Simply, I did not get soluble protein into my hand
Here, I decreased the temperature to two different temperature. 22 and
15 celcius after induction with 0.4mM IPTG. What I found is that my
protein get soluble and I can purify it.
Additionally, I induce my cell when the OD600 reaches the 1.0 or 1.2
Good Luck
Mehmet Sen
hueyjen at gmail.com (Huey-Jen Fang) wrote in message news:<90e43be3.0409161955.1cd4fd26 at posting.google.com>...
> I used two types of GST expression vectors(pET42b and pGEX) to express
> my protein and I had cloned my protein gene in pET42b successfully.
> However, I found that this construction can't express my protein after
> IPTG induction and then I tried to express the pure pET42b vector to
> find out the problem. Unfortunately, I did not observe any GST protein
> expression after induction in BL21, BL21-DE3 and BL21-DE3-pLYS
> strains.If any one had meet the same problem likes my case, please
> give me some suggestions to improve my experiment.
>> Thank You
>> Huey-Jen Fang
