반응 #816419

반응 방정식

kanamycin

chloramphenicol

IPTG

→

Tryptophan

반응 조건

후처리

1. 1
   기타An overnight culture
2. 2
   기타grown to an OD600 ˜0.6 (37° C. with aeration)
3. 3
   기타stored at −80° C. until use
4. 4
   기타Cell debris was removed by centrifugation
5. 5
   추출extract
6. 6
   추출The crude protein extract
7. 7
   여과was filtered
8. 8
   여과filter
9. 9
   세척The column was washed
10. 10
    세척the protein was eluted
11. 11
    농축The desalted protein was concentrated
12. 12
    기타Wild-type alanine racemase was purified

실험 절차

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.

출처