Low expression of pET21a construct - (May/25/2011 )
Hi
I am trying to express an 18kDa protein which I had synthesized and codon optimized for ecoli. It has been successfully inserted into pET21a(+) (confirmed via sequencing and restriction digest) but growth in BL21(DE3) cells has been less than optimal. I am following the protocol we have established in our group for this same protein from different species (mouse, bovine, trout...while this new one is zebrafish): 4 hours growth in LB media with AMP at 37 degrees (OD at least 0.6), induction with 1mM IPTG, grow for another 4 hours at 37 degrees. The SDS PAGE gel shows my protein is there, just not over-expressing (I am going to need a lot - and even after purification it is still just a small band). I have been playing with IPTG concentration (0.5-2.0 mM does not seem to change), induction time (4-6 hours after induction does not change anything), and temperature (O/N induction at 24degrees didn't work).
Any suggestions would be great.
what do you put on a SDS-gel? ...have you fractionized your cells in insoluble and soluble fraction? ...or do you use whole cell lysate?
If your protein is poorly soluble the main content will be in the insoluble fraction in the form of inclusion bodies. It often happens that the protein is poorly soluble and expressed mainly as inclusion body ...and people do believe that there is nearly no expression since the only look at the soluble fraction.
If you protein is expressed but insoluble you can play around with the temperature and the induction strength and time. You can go down to 16°C over night or even 4°C.
Regards,
p
ZF1807 on Wed May 25 17:54:41 2011 said:
Hi
I am trying to express an 18kDa protein which I had synthesized and codon optimized for ecoli. It has been successfully inserted into pET21a(+) (confirmed via sequencing and restriction digest) but growth in BL21(DE3) cells has been less than optimal. I am following the protocol we have established in our group for this same protein from different species (mouse, bovine, trout...while this new one is zebrafish): 4 hours growth in LB media with AMP at 37 degrees (OD at least 0.6), induction with 1mM IPTG, grow for another 4 hours at 37 degrees. The SDS PAGE gel shows my protein is there, just not over-expressing (I am going to need a lot - and even after purification it is still just a small band). I have been playing with IPTG concentration (0.5-2.0 mM does not seem to change), induction time (4-6 hours after induction does not change anything), and temperature (O/N induction at 24degrees didn't work).
Any suggestions would be great.
I normally just run whole cell lysate on the SDS PAGE to confirm protein expression - I am seeing a band at the appropriate size just not the large band I would expect if it was truly overexpressed. I have also purified this protein on a weak anionic exchange column...and run all the fractions coming under UV peaks on an SDS PAGE (these peaks are comparatively low as well but tell me I do have protein coming off the column). Again I see my protein on the SDS PAGE but it is not over-expressed.
what about the growth of your culture? ...does the growth ceases upon induction or do they keep on growing happily?
pDNA on Wed May 25 18:49:13 2011 said:
what about the growth of your culture? ...does the growth ceases upon induction or do they keep on growing happily?
Typically I do not see much of a further increase in OD after induction with IPTG.
hard to say what is going on!
you can try to use a low concentration of IPTG (0.1mM) and try to grow the cells longer (overnight) ...if you can increase the amount of biomass you maybe can compensate for the weak expression.
Regards,
p
ZF1807 on Thu May 26 18:24:59 2011 said:
pDNA on Wed May 25 18:49:13 2011 said:
what about the growth of your culture? ...does the growth ceases upon induction or do they keep on growing happily?
Typically I do not see much of a further increase in OD after induction with IPTG.
It could be that your protein is toxic to the cells. In that case, you need to cut down the induction time. I suggest you try our new bioreactors. Sufficient oxygen makes e.coli cells happier, happier cells will produce more proteins. Please take a look at qizhonglabs.com for more details.