Problem with protein induction - (Jul/20/2018 )

Hi,

I am having an issue with protein induction. I cloned a gene from Poplar into pProxHTa vector and sequenced it completely. But I dont see any induction of protein. I grow the BL21 cells upto 0.8 OD and induce with 0.1, 0.2 and 0.5 mM IPTG.I have tested multiple temperatures after induction (16,20,25,30 C) and multiple time points (1,3,5,16h). I have also cloned the homolog (95% identical) of this gene in Salix and I didn't have any problem in protein induction(0.1 mM IPTG and 20C for 6h). Any suggestions please..

Thanks

Hello, 

After what I read, my first thought was ... he has a mistake in his ORF, but as you sequenced the ORF, this is less likely although you could have acquired mutations in your vector backbone. Did you test several transformants/clones to avoid having a mutation in for instance your promoter ?

You say ... I do not see my protein. Did you try to purify an indication via the His tag? Or do a western blot with an anti-His6 antibody or with the His6 specific colorants of Molecular probes. This to be sure that there are even no minute amounts of protein produced ? 

You can also augment IPTG up to 1mM to have optimal induction.

I would do a first induction at the optimal growth temperature of E.coli (37°c) and not go for lower temperatures ... you can still do this if you have problems with inclusion bodies.

I am also quite astonished  that you have an induction at 20°C of the Salix homolog ... as E. coli is not growing and very inefficiently inducing at this temperature. Try just 37°C ... probably you will get a much better induction.

Best

MaKo on Mon Jul 23 17:02:23 2018 said:

Hello, 

 

After what I read, my first thought was ... he has a mistake in his ORF, but as you sequenced the ORF, this is less likely although you could have acquired mutations in your vector backbone. Did you test several transformants/clones to avoid having a mutation in for instance your promoter ?

 

You say ... I do not see my protein. Did you try to purify an indication via the His tag? Or do a western blot with an anti-His6 antibody or with the His6 specific colorants of Molecular probes. This to be sure that there are even no minute amounts of protein produced ? 

 

You can also augment IPTG up to 1mM to have optimal induction.

 

I would do a first induction at the optimal growth temperature of E.coli (37°c) and not go for lower temperatures ... you can still do this if you have problems with inclusion bodies.

 

I am also quite astonished  that you have an induction at 20°C of the Salix homolog ... as E. coli is not growing and very inefficiently inducing at this temperature. Try just 37°C ... probably you will get a much better induction.

Best

HI,

Thanks for the response. I have used the same vector for my cloning of the Salix homolog, but it is likely as you have pointed out there might mutations in my vector backbone. I have sequenced 2 clones and both have the same issue, no induction upon IPTG treatment.

As I was able to see stronger induction of protein in my other samples I haven't ventured into western blotting yet.

I grow the cells upto 0.8OD at 37C and then induce with IPTG at lower temperatures.

WIll give your suggestion a try and get back.

Many thanks again