SeCYS


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Protocol

The protocol for labelling is simple, achieves high level of biosynthetic incorporations and could be applied to any sulphur containing protein, even those containing disulphide bridges.

The main drawbacks of Se-Cys labelling are the alteration of the protein solubility and the formation of inclusion bodies.


The protocole for SeMet-SeCys double labeling is based on the use of an auxotrophic cys - E. coli strain and inhibition of the methionine biosynthesis pathway;
it therefore requires a defined medium (with high concentration of isoleucine, lysine, and threonine).

The protocol can be used in either fermentors (up to 10L) or shake flasks, as long as the change of medium is performed under sterile conditions.
For volumes up to 100mL, the fermentation is best carried out in sterile centrifugation bottles.

Materials

Cysteine auxotrophic host strain:

E. coli BL21(DE3) selB::kan cys51E

In this strain sel B is inactivated by the insertion of a kanamycin resistance cassette. The cell requires cysteine/cystine.

Expression vector containing the gene coding for the protein of interest
Medium
DSMM and DSeMM (see recipe)
Wash buffer
LB

Host cell transformation

1. Transform the expression vector containing the gene of interest into the appropriated cysteine auxotrophic host cell.

The efficiency of the labelling procedure relies on a very low basal expression of cloned target genes during the growth phase. The T7 polymerase based pET system (and the matching host cell) provides such an efficient and stringently controlled expression of the target protein. Additional control of transcription can be achieved via the constitutively expressed T7 lysozyme gene carried by the pLysS plasmid. Nevertheless other plasmids have been successfully used when associated with the suitable host cell.

2. Test for expression.


Fermentation

3. Prepare and sterile filter the DSM and DSeM media, and sterilise either the fermentors or the shake flasks as well as the centrifuge bottles.

4. Grow an overnight culture, corresponding to 1/10 of the fermentation, in LB medium at 37C. Centrifuge at 3000 g and wash two times with wash buffer.

Use the antibiotics necessary for plasmid selection as well as those essential to maintain auxotrophy of the host cells.

5. Resuspend the cell pellet in DSM medium and transfer to either the fermentor or a shake flask.

The carbon source can be either glycerol or glucose.

6. Grow the cells to an OD600nm of 3-4 and induce with 1 mM IPTG. After 10 min induction, add 10 mg/L of chloramphenicol and keep on for 5 min before harvesting the cells, under sterile conditions, by centrifugation.

Addition of chloramphenicol blocks protein synthesis and hence inhibits chromosomal replication whilst producing plasmid in high copy number.

7. The cell pellet is washed twice with wash buffer and resuspended in DSeM medium containing 1mM IPTG.

When a T7 polymerase expression system is used, best results are obtained with the addition of the E. coli polymerase inhibitor, rifampicin, to a final concentration of 400 mg/L.

8. Monitor protein expression and harvest the cells accordingly.

REAGENTS AND SOLUTIONS

Use Milli-Q-purified water or equivalent for the preparation of all solutions and media.

DSM medium
10 g K
2HPO4, 3H2O
2 g NH
4Cl
1 g sodium acetate
2.75 g sodium succinate
0.435 g magnesium acetate, 4H
2O
0.01 g calcium chloride, 2H
2
O
1.6 g serine
1.0 g leucine
0.4 g alanine
0.4 g sodium glutamate, H
2O
0.4 g glutamine
0.4 g arginine, HCl
0.4 g glycine
0.25 g aspartic acid
0.1 g asparagine
0.1 g histidine, HCl, H
2O
0.1 g lysine, HCl
0.1 g proline
0.1 g threonine
0.1 g tyrosine
0.1 g isoleucine
0.05 g tryptophan
0.05 g valine
0.05 g phenylalanine
0.05 g cysteine
0.05 g methionine
10 g glycerol or glucose
40 nM boric acid*
3 nM cobalt chloride*
0.1 nM copper chloride*
8 nM manganese chloride*
1 nM zinc chloride*
100 mg nicotinamide
50 mg thiamine
0.5 mg biotin
H
2O to 1 litre

* Sodium glutamate is dissolved in water, tyrosine and aspartic acid are dissolved in 0.1N NaOH whilst the other 17 amino acids are dissolved in 0.1N HCl prior to sterile filtration through a 0.22 m membrane.
* The carbon and nitrogen sources are prepared as 50% and 20% stock solutions respectively and sterile filtered.
* The buffers are prepared as 10x solutions and heat sterilised and the salts are 200x solutions sterile filtered.
* The trace elements* are prepared as a 10,000x solution and the nicotinamide solution and the biotin-d6/thiamine mix are at 1000x and 250x respectively and sterile filtered.
* The stock solutions are stored at room temperature till used.

Mix each ingredient and add water to 1 liter. Final pH should be 7.0-7.3 (adjust with NaOH as needed).
Antibiotics should be added (if appropriated) just before use: 

kanamycin (15mg/L), carbenicillin (100 mg/L).



DSeM medium

The DSeM medium is similar to the DSM medium but methionine and cysteine are replaced by Seleno-D/L methionine and Seleno-D/L cystine at concentration of 0.4
mM and 0.6 mM, respectively.

Wash buffer

> The buffer is heat sterilized.

10 g K2HPO4, 3H2O
1 g sodium acetate
H
2O to 1 liter.





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