Reaktion #816419

Reaktionsgleichung

kanamycin

chloramphenicol

IPTG

→

Tryptophan

Reaktionsbedingungen

Aufarbeitung

1. 1
   SonstigeAn overnight culture
2. 2
   Sonstigegrown to an OD600 ˜0.6 (37° C. with aeration)
3. 3
   Sonstigestored at −80° C. until use
4. 4
   SonstigeCell debris was removed by centrifugation
5. 5
   Extraktionextract
6. 6
   ExtraktionThe crude protein extract
7. 7
   Filtrationwas filtered
8. 8
   Filtrationfilter
9. 9
   WaschenThe column was washed
10. 10
    Waschenthe protein was eluted
11. 11
    EinengenThe desalted protein was concentrated
12. 12
    SonstigeWild-type alanine racemase was purified

Vorschrift

An overnight culture with the pET30Trp racemase construct was subcultured into fresh LB medium with the appropriate antibiotics (50 μg/ml kanamycin and 20 μg/ml chloramphenicol) and grown to an OD600 ˜0.6 (37° C. with aeration). Expression was induced with 100 μM IPTG and incubation was continued at 37° C. with aeration for 2 hours. The cells were harvested by centrifugation and stored at −80° C. until use. The cell pellet was thawed on ice and cells were lysed using BugBuster Primary Amine Free Cell Lysis Reagent and Benzoase Nuclease (Novagen, Madison, Wis.). Cell debris was removed by centrifugation and the supernatant was used as the crude protein extract. The crude protein extract was filtered using a 0.45 μm syringe filter and applied to a HisBind column (Novagen, Madison, Wis.) that had been pre-equilibrated according to the manufacturer's instructions. The column was washed and the protein was eluted as directed in the manufacturer's protocol. The purified protein was desalted with a PD-10 column (GE Healthcare, Piscataway, N.J.) using 50 mM potassium phosphate pH 8.0, 10 μM pyridoxal-5′-phosphate (“PLP”) as the eluent. The desalted protein was concentrated using Amicon centrifugal concentrators (Millipore, Billerica, Mass.). Wild-type alanine racemase was purified as described above.

Quelle