IMPACT I (intein system)

Robin Lee Tremblay a69rlt at morgan.ucs.mun.ca
Wed Aug 20 18:16:43 EST 1997


Has anybody tried to use the intein system? I'm having a problem with in
vivo cleavage. Every time I run a western using the antibody against the
intein I end up with two bands - one with my protein and one corresponding
to the intein and chitin inding domain. I tried inducing at 13, 30 and
37C, and still have the problem (induction didn't work at 13C)

I also tried using BL21 and MC1061 strains to induce the protein and got
very different induction results. The BL21 does not give nice induction
and the MC1061 looks like it allows induction to proceed but has the
cleavage problem. My protein ends with a tyrosine, but has two arginines
in the C terminal area. I used the SapI site so I don't have any extra
amino acids hanging off the end of the protein and would like to avoid
putting any extra amino acids on. 

Any suggestions anyone has about using a new strain or finding a different
way to stop cleavage would be welcome. Thanks in advance.  


Robin
--
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					|	
Robin Tremblay				|	You can't always be young
a69rlt at morgan.ucs.mun.ca		|	 
Department of Biochemistry		|	But you can be immature	   
Memorial University of Newfoundland	|		forever!
(709)-737-2538				|	
					|	:}
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