Se-Met protein expression

Kurt S. Thorn ksthorn at tucson.princeton.edu
Tue Mar 26 16:58:48 EST 1996


In article <4j93as$lv6 at netnews.upenn.edu>,
Satish Nair <satish at a.chem.upenn.edu> wrote:
>In article <4j7lo7$ij0 at lll-winken.llnl.gov> rupp1 at llnl.gov writes:
>>Hi to all bug-people :
>>
>>I wonder whether someone could give me some leads about how to express proteins 
>>with Se-Met for MAD phasing. I read the review by Ms. Doublie and Charlie Carter but
>>that's it about.  In particular, I'd like to know about suitable bug strains, new reviews and recipes, and
>
>Since you brought this up... I've been told that it's posssible to make
>BL21(DE3) into a methionine auxotroph by spiking the growth media with
>amino acids which inhibit methionine synthesis.  Wouldn't it then be possible
>to incorporate Se-Met into BL21(DE3) expressed proteins or are there
>other complications?

I have recently used this procedure to produce SeMet protein.  You just grow
up your bacteria in M9 minimal media, and add lys, phe, thr, ile, leu, val,
and SeMet 15 min before induction.  Mass spec indicated substitution; we have
synchrotron time starting tomorrow to try the MAD work.

The reference is: Van Duyne et al. JMB 229:105-124, 1993.
There is also a forthcoming paper by Doublie in Meth. Enzymol.

If you have questions or want related references, drop me an email.

Kurt

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