Expression and purification of TEV protease
The plasmid (pSF455) encodes a modified TEV as a fusion protein with MBP in pQE80N
(MBP-tev-H14-TEV(CDS)-Arg5). The original TEV contains a self-cleavage site
that has been removed by S Frey. The Arg tail served for better purification by
an ion exchange step. The MBT is cleaved off in vivo.
Expression
- strain: E. coli BL21(DE3) star rosetta
- medium: TB, 0.8% gycerol, 2 mM MgCl2, 300 µg/ml
ampicillin, 34 mg/ml chloramphenicol
- induction: with 0.1 mM IPTG at OD=0.6, overnight at 18-20¡C
- collect cells and suspend in extraction buffer with 1 mM PMSF at 100 OD/ml.
- freeze in liquid nitrogen
- thaw the cells, add beta mercaptoethaol to 10 mM (stock is 14.3 M)
- lyse by sonication: 3 x 2 min, max output (10), 40% duty cycle on ice
- spin in ultacentrifuge, 40 k, 4¡C
- collect the supernatant
Purification by Ni chromatography
- prepare the Ni sepharose column, 4 ml
- equilibrate the column with extraction buffer (3 volumes)
- apply the cleared lysate, collect the flow-through
- wash with 2x 10 ml extraction buffer + 5 mM DTT
- elute with elution buffer, collect 1 ml fractions
- analyse the fraction by amidoblack staining and SDS-PAGE
- pool appropriate fraction
- change the buffer to storage buffer by gelfiltration with a PD-10
colum or a desalting column
- test the activity of the TEV
Buffer:
extraction buffer:
50 mM K-PO4 [pH 7.5], 300 mM NaCl, 1 mM Imidazol
elution buffer:
extraction buffer, 300 mM imidazol
storage buffer:
20 mM Na-phosphate [pH 7], 50 mM NaCl, 0.1 mM EDTA, 1 mM DTT, 10% glycerol