Radiolabelling proteins overexpressed in E.coli

user unknown szsclark at chip.ucdavis.edu
Fri Nov 18 18:51:22 EST 1994


Dear Netters,

I am overexpressing a smallish protein (ca. 20 kD) in e. coli using the
Novagene pEt vectors in BL21/DE3 (pLysS).  My protein expresses quite well
and i get yields of several hundred micrograms from my preps.  My problem
is the specific activity of the protein.  I am labelling the protein with
3H-leucine.  For various reasons I can't use other radiolabels and 3H is
best for my purposes, but theoretical calculations of the potential
specific activity achievable if all the leucines in my protein are 3H'd
are about 1000x what I actually get.  The specific activities I am
achieving are within the range reported by other people who have expressed
similar proteins so I don't think there is anything 'wrong' with my
protocol, which is pretty standard.  I am wondering if there is some
mechanism which prevents the 3H-leu being incorporated into every
position, eg. competition with an intracellular pool of leucine that is
synthesized by the coli?  Is 3H -leucine not taken up by the coli as
efficiently as non-3H amino acids?  If I increase the amount of 3H-leucine
I add to the prep, the specific activities appear to increase
proportionally, suggesting the available 3H-leucine is non-saturating.  Or
is there something I can do to improve my protocol and my specific
activities?  I haven't been able to find any suggestions in the literature.  
Your suggestions are welcome. 

My abbreviated protocol is;

Grow fresh cells in M9 + Bactotryptone + Carbenicillin (100 ug/ml) until 
OD600 ca. 0.3

Wash in M9, resuspend to 1/3 rd vol. in M9+Carb+Chloramphenicol. Store 
o/n at 4oC. (I titre here to check plasmid stability)

Inoc. 300 ml fresh M9+carb+chlor with 6 ml of starter culture. Grow to 
OD600 =0.2.

Wash cells 2x in M9. Resusp. in 1/10th vol of M9+C+C. Add IPTG to 0.4 mM. 

Grow at 37oC for 40 min.

Add 10 ml of culture to flask containing 1.5 mCi of dried 3H-leucine. 
Grow for 4 to 5 hours.

spin down cells, resuspend in 10ml of 20mM Tris 7.5, 20% sucrose, 1mM EDTA. 
Incubate 10 min on ice.  

Spin down cells, resuspend in 5ml of ice-cold H2O with 1mM PMSF, 20 ug/ml 
aprotinin, 1 ug/ml leupeptin. Incubate 10 min on ice. Add equal vol 2 
PBS. Freeze overnight.

Lyse by probe sonication. Wash inclusion bodies 3x with 1xPBS, 5 mM EDTA, 
25% Sucrose, 1% triton X100. wash 3x with cold H2O.

I usually get between 200 and 300 ug of protein of between 600, 000 and 1 
x 10e6 dpm/ug.

thanks for reading all this!  any suggestions welcomed.

Sonya Clark



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