*From some articles that I was reading. Does not summarize all the P. horikoshii procedures.
1) Desai, Kevin K., and Ronald T. Raines. "tRNA Ligase Catalyzes the GTP-dependent Ligation of RNA with 3'-Phosphate and 5'-Hydroxyl Termini." Biochemistry including Biophysical Chemistry and Molecular Biology 51 (2012): 1333-335.
· Expressed for 2 hrs at 32C. Spun down.
· Resuspended in buffer A (50 mM Tris-HCl, pH 7.7, 300 mM NaCl, 0.5 mM DTT) w/ 25 mM imidazole
· Lysed with a French press and centrifuged.
· Super added to a Ni-NTA column equilibrated with buffer A w/ 25 mM imidazole.
· Column washed with 10 column volumes buffer A w/ 25 mM imidazole.
· Column washed with 10 column volumes buffer A w/ 40 mM imidazole.
· Eluted with buffer A containing 250 mM imidazole.
· Dialyzed against 2L of 20 mM Tris-HCl, pH 7.4, 200 mM NaCl (note no DTT).
Comments: No other details given (such as how many mg/ml was collected, T at which it was conducted, protease used). Expression was only done for 2 hrs. Two buffers. No DTT in final. Purification of P. horikoshii homologue was conducted exactly as described for E.coli rtcB except that its stability required a higher salt concentration and was dialyzed against 20 mM Tris-HCl buffer, pH 7.4, 300 mM NaCl.
2) Tanaka, Naoko, and Stewart Shuman. "RtcB Is the RNA Ligase Component of an Escherichia Coli RNA Repair Operon." Journal of Biological Chemistry (2011): 7727-7731.
· Expressed for 16 hrs at 17C. Spun down. Rest of procedure performed at 4C.
· Resuspended in 50 ml buffer A (50 mM Tris-HCl, pH 7.4, 250 mM NaCl, 10% sucrose)
· Lysozyme added to 0.2 mg/ml.
· After mixing 30 min, suspension was adjusted to 0.1% Triton X-100 and incubated 15 min.
· Sonication and centrifugation.
· Lysate mixed 1 hr with 12 ml 50% Ni-NTA slurry equilibrated in buffer A.
· Resin recovered by centrifugation and resuspended in 25 ml buffer B (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 10% glycerol) with 25 mM imidazole.
· Centrifugation and resuspension of resin in buffer B repeated 3x, then poured into column.
· Column washed with 25 ml of 2M KCl in 50 mM Tris-HCl, pH 7.4.
· Eluted stepwise with 7-ml aliquots of 100, 200, 300, 400 and 500 mM imidazole in buffer B.
· (Discussion of removal of the His10Smt3 tag.)
· Tag-free rtcB prep concentrated by centrifugal ultrafiltration to 16 mg/ml (in 500 ul).
· Gel-filtered through a 120 ml 16/60 HiLoad Superdex 200 pg column equilibrated w/ buffer C (10 mM Tris-HCl, pH 8.0, 350 mM NaCl 1 mM DTT) at flow rate of 1.0 ml/min, collecting 2-ml fractions.
· Peak rtcB fractions pooled and concentrated via centrifugal ultrafiltration.
· Final yield = 5 mg from 1-L culture.
Comments: Very detailed. 16y hrs expression. High protein yield 16 mg/ml (in 500 ul), 5 mg total yield from 1 L. All conducted at 4C. Three buffers. No glycerol in final; uses DTT; pH is higher.
3) Desai, Kevin K., Craig A. Bingman, George N. Phillips, Jr., and Ronald T. Raines. "Structures of the Noncanonical RNA Ligase RtcB Reveal the Mechanism of Histidine Guanylylation." Biochemistry including Biophysical Chemistry and Molecular Biology 52 (2013): 2518-525.
Procedure for purifying rtcB from P. horikoshii
Comments: This protein had no his-tag, perhaps necessitating some but not all of the changes.
· Expressed 3 hrs.
· Resuspended in 8 ml per gram of wet pellet in buffer A (50 mM MES-NaOH pH 5.6, 45 mM NaCl, 1 mM DTT)
· Cell lysis and centrifugation.
· Lysate loaded onto a 5 ml HiTrap HP SP cation-exchange column.
· Column was washed with 25 ml buffer A.
· rtcB eluted w/ NaCl gradient of buffer A (45 mM to 1.0 M) over 20 column volumes.
· Fractions containing rtcB dialyzed o/n @ 4C against 4L buffer A.
· Dialyzed rtcB loaded onto 5 ml HiTrap heparin column.
· Purified rtcB eluted as above.
· Purified rtcB dialyzed o/n @ 4C against 4L of 10 mM HEPES-NaOH, pH 7.5, 200 mM NaCl
Major Differences from Previous
· Grown 3 hrs
· Buffers differ
· Bacterial proteins precipitated and removed by incubating lysate at 70C (additional centrifuge)
· Cation exchange column used (since no his tag)
· Final buffer = HEPES-NaOH, rather than Tris-HCl
4) Englert, Markus, Shuangluo Xia, Chiaki Okada, Akiyoshi Nakamura, Ved Tanavde, Min Yao, Soo Hyun Eom, William H. Konigsberg, Dieter Soll, and Jimin Wang. "Structural And Mechanistic Insights Into Guanylylation of RNA Splicing Ligase RtcB Joining RNA Between 3′ -terminal Phosphate and 5′-OH." PNAS 109, 38 (2012): 15235-15240.
· Grown to 0.8 OD. Expressed at 18C for 16 hrs.
· Harvested cells were disrupted in Ni-NTA lysis buffer (20 mM Tris-HCl, pH 8, 500 mM NaCl, 3mM MnCl2, 10 mM imidazole, 10 mM B-ME).
· Final buffer = 10 mM Tris-HCl, pH 8, 200 mM NaCl, 3 mM MnCl2, 10 mM B-ME.