CryoCrystallography

---

 

Order from Molecular Dimensions Ltd. 

Unit 7, Acorn Business Centre, Oaks Drive, Newmarket, CB8 7SY 

Tel: [phone]   Fax: [phone] 

 

SelenoMet™ Medium

 

 

Instructions for Use

 



 

Based on a synthetic M9 minimal media 

supplemented with glucose, vitamins and 

amino acids with the exception of L-

methionine. (1,2)

 

 

The medium will be yellow when fully 

reconstituted.  Bacterial growth can be carried 

out either in the presence of L-methionine or 

L-selenomethionine (SeMet).  Plasmid 

expression constructs should be transformed 

into a suitable methionine requiring 

auxotrophic strain such as 

E. coli 834(DE3) if 

using the T7 polymerase based pET system.  

We have not tried the media out using 

methods to inhibit methionine biosynthesis in 

heterotrophic strains but in principle this 

media could be modified to achieve this. 

General Growth Considerations. 

The growth and induction conditions will vary 

depending upon the vector system and protein 

to be expressed.  To combat poor growth 

rates Ramakrishnan and Graziano have used 

cultures in the mid log phase of growth to 

innoculate into larger culture volumes for the 

induction of expression.  Other investigators 

have grown starter cultures in minimal 

medium containing L-methionine followed by 

extensive washing of the cells before 

innoculating into L-SeMet containing media. 

Dissolve 21.6 g of SelenoMet Medium Base in 

1 litre of DI water and autoclave,  

Dissolve 5.1g SelenoMet Nutrient Mix in 50 ml 

of DI water and sterile filter and add to base 

medium to make up methionine minus 

medium. 

The Methionine and SelenoMethinone 

solutions are made up as 250x concentrates, 

so 4ml should be added per litre of medium. 

At Daresbury (in collaboration with Ian Cummins 

and Robert Edwards of Durham University), the 

SelenoMet media has been used to express and 

SeMet-label an 

Arabidopsis hydrolase expressed in E. 

coli  834(DE3), using pET24a, as a C-terminal 

polyhistidine-tagged protein.  A 100 ml culture was 

grown overnight at 37

0

C containing L-methionine, 

the cells were then pelleted, washed 3 times in 100 

ml  of  sterile  water,  resuspended  in  1  ml  of  water 

and innoculated into a litre of prewarmed (30

0

C) 

minimal media containing L-SeMet.   

The cells were grown for 2 hours before the addition 

of IPTG to 1 mM and growth was continued for a 

further 6 hours prior to cell harvest.  Following 

nickel affinity chromatography a yield of at least 35 

mg per litre was achieved with SeMet-labelling levels 

of >99% as judged by electrospray mass 

spectrometry.  Comparable yields of SeMet-labelled 

protein were observed using either media prepared 

in the laboratory or with the commercial formulation.  

The SeMet-labelled protein crystallised under the 

same conditions as the methionine-containing 

protein  

Molecular Dimensions gratefully acknowledge 

assistance from Tony Fordham-Skelton and 

Katherine McAuley, Biology and Medicine College, 

Department of Synchrotron Radiation, CLRC, 

Daresbury Laboratory, Warrington, UK., in testing 

the media fomulation and preparing the instructions 

for use. 

1. Ramakrishnan and Graziano; http://alf1.mrc-

lmb.cam.ac.uk/~ramak/madms/segrowth.html 

2. Ramakrishnan V., Finch J.T., Graziano V., Lee 

P.L., Sweet R.M. Nature 362, 219, (1993) 

 

 

Order information 

MD12-500

 

SelenoMet Medium Complete 

MD12-501

 

SelenoMet base media and nutrients 

MD12-503

 

SelenoMethionine solution 250x 

MD12-504

 

Methionine solution 250x