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pET47b proteins not expressing in BL21 (DE3) - (Aug/22/2011 )

Hi there,

I have a gene for a 25 kDa bacterial metallo protein placed into pET47b / BL21 (DE3). I've done an expression trial and I can't see any bands for expression-- what are some of the options I can do to play around with to see if it can express?

-Luria Bertani-

have you checked the construct if everything is in frame and free of mutations prior to expression?

if you want help you have to give more details on your expression experiment e.g.
- IPTG concentration used
- duration of expression after induction
- Optical density at induction
- course of OD after induction (growth/no growth)
- confirmation expression (PAGE, western blot, ...?)

if you give us more details we can give you suggestions!

Regards,
p

-pDNA-

Hi pDNA, thanks for your reply.

This was an expression test, the construct itself seems fine and has been sequenced. The gene is under T7 promoter control.
The IPTG concentrations used were 0mM, 0.1 mM, 0.5 mM, 1 mM
Induction took place when the OD600 reached 0.5 (I didn't measure the OD after induction)
The duration of the expression was 24 hours with samples taken at 3 hours, 6 hours, 12 hours and 24 hours after induction
SDS-PAGE was used to check for any bands that might appear- none seem to jump out

-Luria Bertani-

Hola, As always I say I think that the problem is the host.It hasn´t any control to repress any basal expression which could give the extinction of producers recombinant bacteria at first stages of culture , surviving no producers . This is noticed when early growth phases are too long.
Whit this host I would change tryptone by soytone, and add a low glucose (o.1-0.2 g/l) concentration from the seed. If the problem isn´t solved I woud change to any LacIq strain, or better to any lac- lacIq strain as Origami (DE3 lacI) or better OrigamiB DE3 placI , LacI of the plasmid isn´t enought to repress early expression. Cross your fingers and buena suerte

-protolder-

what antibiotic? ...Ampicillin?

Regards,
p

-pDNA-

Hola Peter Sorry by my bad expression, I mean add glucose from the preinoculum. Not always glucose repress expression but sometimes it helps (uv5 promoter isn´t sensible at glucose). Other possibility is using as preinoculum all the colonies of a fresh transformation whit pure plasmid and follow the expression from the begining withouth induction and induce with lactose o0.5 g/l. buena suerte

-protolder-