Purification of recombinant T7 RNA polymerase

A tagged version of T7 RNA polymerase with a His-tag (C- or N-terminal is not clear) is encoded by the plasmid pT7_911 (AmpR). The identiy of the plasmid is unknown. It is possibly a pQE derivative that does not require BL21(DE) host strains. The plasmid was obtained from M Boutros/DKFZ but originates from a person in M Mlodzik's lab in NY.

Expression of recombinant His-T7 RNA pol

  • Transform competent E. coli (DH5a or BL21) with the plasmid pT7_911. Inocculate 100 ml of LB/Amp with 900 ul of the suspension. Spread the remaining 100 ul on a LB/Amp plate. Grow the preculture overnight.
  • Inocculate 1 l LB/Amp (prewormed to 37¡C) with 50 ml of the pre-culture. Grow to an OD600 of about 0.6-0.8. Use a big flask
  • Add IPTG to c=0.4 mM to induce expression.
  • Measure the OD600, collect the cells, suspend in lysis buffer (2-3 ml per g wet weight)
  • Freeze the suspension or immediately proceed to purification

Purification of His tagged T7 RNA pol

Purify the protein by metal affinity purification with chromatography or by a batch procedure.
Pool the fractions with protein.
Exchange the elution buffer with storage buffer by gel filtration with a NAP-10 column. In case of gel filtration use a 1x buffer without glycerol. Add the glycerol after chromatography and complement the beta-ME to 1x.

Storage buffer

    10 mM K-phosphate pH 7.9
    200 mM KCl
    0.1 mM EDTA
    30 mM beta-mercaptoethanol
    0,1% Tween20
    50% glycerol
Precipitates form in a 2x storage buffer (without the glycerol) at 4¡C.

Quality control of the preparation

Measure the protein concentration by Bradford in comparision to a BAS standard.
Compare the activity of the RNA polymerase to a commerical preparation.
Prepare/obtain a bidirectional T7 template by PCR with primers introducing T7 sites
on both ends. Synthesize dsRNA with a bidirectional template. dsRNA is stable in comparision to single-stranded RNA, wich makes the analysis more robust.
Purify the product by phenol extraction
Determine yield by measuring the absorption at 260 or by gel electrophoresis.

Reference

He B, Rong M, Lyakhov D, Gartenstein H, Diaz G, Castagna R, McAllister WT, Durbin RK. Protein Expr Purif. 9 (1997) 142Ð151

Purification: A. Frank-10/08

After Ni chromatography two bands at 98, which corresponds to the band of the enzyme from Roche, and below 90 kd were obtained. The activity was comparable to the enzyme from Roche. The enzyme is stored at -20¡C in 50% glycerol. Please find here the documentation of the purification.