Protein expression - decreased expression (Oct/05/2006 )
Hi everyone,
i have a mycobacterial gene cloned in pRSET A vector under T7 promoter. There was very good expression at 0.3mM IPTG induction for 3hours since the last 5-7 years. But recently we are finding
that the expression is decreasing a lot and even the activity of the purified enzyme. Can some one
please suggest me why could this be happening and possible ways to troubleshoot since my whole work of mutants is based on the activity of the wild type enzyme
hoping suggestions at the earliest
thanking all in advance
Leelaram
-leelaram-
QUOTE (leelaram @ Oct 6 2006, 03:51 AM)
Hi everyone,
i have a mycobacterial gene cloned in pRSET A vector under T7 promoter. There was very good expression at 0.3mM IPTG induction for 3hours since the last 5-7 years. But recently we are finding
that the expression is decreasing a lot and even the activity of the purified enzyme. Can some one
please suggest me why could this be happening and possible ways to troubleshoot since my whole work of mutants is based on the activity of the wild type enzyme
hoping suggestions at the earliest
thanking all in advance
Leelaram
i have a mycobacterial gene cloned in pRSET A vector under T7 promoter. There was very good expression at 0.3mM IPTG induction for 3hours since the last 5-7 years. But recently we are finding
that the expression is decreasing a lot and even the activity of the purified enzyme. Can some one
please suggest me why could this be happening and possible ways to troubleshoot since my whole work of mutants is based on the activity of the wild type enzyme
hoping suggestions at the earliest
thanking all in advance
Leelaram
To find the reason could be very difficult:
- Different or new medium, mayby variation in the salt concentration (high or low salt)
- starting with a fresh preculture?
- Problems with your antibiotics, wrong ammount?
- We had a problem with the detergents in the dishwasher (no joke), half a year no expression!!!
- Temperature-problem, to high temperature leads to inactive protein and low concentration in the supernatant
- wrong cells
- fresh or old IPTG solution or wrong concentration (sorry, nobody wants to hear this...)
...
Sorry, but this is a bad problem to solve...
-ms-olli-
QUOTE (leelaram @ Oct 6 2006, 06:51 AM)
Hi everyone,
i have a mycobacterial gene cloned in pRSET A vector under T7 promoter. There was very good expression at 0.3mM IPTG induction for 3hours since the last 5-7 years. But recently we are finding
that the expression is decreasing a lot and even the activity of the purified enzyme. Can some one
please suggest me why could this be happening and possible ways to troubleshoot since my whole work of mutants is based on the activity of the wild type enzyme
hoping suggestions at the earliest
thanking all in advance
Leelaram
i have a mycobacterial gene cloned in pRSET A vector under T7 promoter. There was very good expression at 0.3mM IPTG induction for 3hours since the last 5-7 years. But recently we are finding
that the expression is decreasing a lot and even the activity of the purified enzyme. Can some one
please suggest me why could this be happening and possible ways to troubleshoot since my whole work of mutants is based on the activity of the wild type enzyme
hoping suggestions at the earliest
thanking all in advance
Leelaram
I suggest you also have your construct sequenced (again) to make sure that it hasn't been mutated. I had a similar problem with a different protein (expressed from the same vector) that appear to be toxic to the cells, and it turned out to be a mutated version that wasn't expressed in E.coli.
-mofe-