protein expression in BL21

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tkoshy
tkoshy's picture
protein expression in BL21

Hi,
I am trying to get protein expression in BL21 cells. I am working on plasmodium protein EMP1, cloned in pQE30. The clone is correct but i am not getting any protein expression in BL21. I am usiing 1mM IPTG & even 0.5mM but same result. The insert size is 1341bp, protein is 49.7kd.

Ivan Delgado
Ivan Delgado's picture
 

 
Hi tkoshy,
Have you tried to grow yours cells at 28oC? While 37oC is the standard growth temperature, in some cases growing your cells at 28oC (slower growth) can dramatically increase the amount of protein that is soluble vs insoluble. Which brings me to another point: did you check to see if there was any insoluble protein in your extracts?
Another consideration is the amount of time you induce. Sometimes proteins are unstable so you should also grow your culture for 2-4 hours after induction. Other times an overnight induction may work too (if the protein is unstable and the expression is not very robust).
Good luck
 

Chin Fen Teo
Chin Fen Teo's picture
 Hi there, I agree with Ivan

 Hi there, I agree with Ivan on lowering the temperature. I used to overexpressed Plasmodium proteins in a lab that I rotated many years ago, and I had to grow them at around 16 degree for overnight... If not, try to see whether the MagicMedia from invitrogen can help with your problems....

tkoshy
tkoshy's picture
Hi,

Hi,
I have tried inducing the cells at lower temp (25 degree) for overnght, but still the same result. The result which I get are not convincing, actually I get a band in the -IPTG sample as well.I carried on a western but the result was weired maybe because the anti-his antibody we are using is not good. I also want to share another set of info. that when I try to transform 50ul BL21 with 2ul construct using I never get any colonies instead I have to use 200ul of BL21.

Chin Fen Teo
Chin Fen Teo's picture
 Well, if the protein

 Well, if the protein expression is leaky, it is possible to detect at low amount even before adding IPTG... Have you checked the plasmid that obtained from the transformation? It sounds to me that your transformation efficiency is really low... And in my hands, usually it implies that the bacteria didn't really pick up the plasmid... Miniprep and restriction digest to see whether it is there... Also, you might want to try different fusion (says, using MBP instead of His) ? From what I understand is that not all protein prefer His epitope... 

varsha
varsha's picture
Hi tkoshy.

Hi tkoshy.
My experience with some of the Plasmodium proteins was to grow the transformants at lower temperature. I also had leaky expression i.e expression in -IPTG sample. I ended up optimizing the grwoth condition  for 26C and 0.2 mM IPTG for a GST fusion protein. Another protein did not express very well and my colleague had to use a codon optimized strain to E. coli for expression. Unfortunately, I donot  remember which strain was used.  You may want to check what other Plasmodium labs use for protein expression?
You should try a good, fresh batch of BL21 cells and optimize the expression condition. 
If that does not work try to find may codon optimized strain.
Good luck!
Varsha

dogramicrobiology
dogramicrobiology's picture
hi

hi
 it is very difficult to geting any protein cloned in pQE vector to express in in BL 21 cells. for pQE we require M 15 Cells instead of BL 21 cells. try with M 15 CELLS  

 Good luck