Protocol H.1

Expression & Purification of His Tagged Proteins in E. coli

This protocol is essentially as described by Qiagen who is a good source for the Ni-NTA agarose resin.

Solutions

Sonication Buffer

50 mM NaPO4 pH 7.8 0.59 g monobasic NaH2PO4

12.26 g dibasic Na2HPO4

300 mM NaCl 17.5 g NaCl up to 1 liter with Q

Wash Buffer

50 mM NaPO4 pH 6.0 6.05 g monobasic NaH2PO4

1.64 g dibasic Na2HPO4

300 mM NaCl 7.5 g NaCl

10% glycerol 100 ml glycerol

up to 1 liter with Q

 

Elution Buffer

500 mM immidazole 0.34 g immidazole

up to 10 ml with Wash Buffer

Dialysis Buffer

20 mM Hepes 7.9 20 ml 1M Hepes pH 7.9

10 mM KCl 0.75 g KCl

10% glycerol 100 ml glycerol

0.5 mM DTT 1 ml 0.5 M DTT

1 mM PMSF 10 ml 100 mM PMSF

 

 

Procedure

 

• Transform the His tagged construct (PRSET or derivatives) into the E. coli strain BL21 DE3 pLys and Plate on LB plates containing ampicillin and chloramphenicol.

• Restreak a single colony on LB Amp, Cm and grow overnight. Plug a single colony in 100 ml LB Amp, Cm and grow overnight.

• Innoculate 500 ml LB Amp, Cm with 50 ml of this fresh overnight culture. Take the OD600 and monitor every hour until the OD is between 0.6 and 0.9.

• Add 10 ml 100 mM IPTG and additional Amp and Cm. Grow for 3 hours and add an additional 10ml 100 mM IPTG along with Amp and Cm.

• After an additional 3 hours of growth harvest the cells by spinning in the Sorval GSA rotor for 5 minutes at 6,000 rpms. Resuspend the pellet in sonication buffer (10 ml for every 0.1 g wet weight). Approximately 12.5 ml for every 250 ml culture.

• Freeze overnight and thaw the next day on ice. Sonicate on 80% duty cycle set on 6 with the microtip for 30-40 bursts using a Branson sonifier. Be careful not to overheat the sample. Spin at 4° for 10 minutes to pellet the debris.

• Equilibrate the Ni-NTA Agarose resin in Sonication Buffer and add 50-100 ml of a 50% slurry per ml of sonicate. Tilt for 30 minutes at 4°.

• Wash 4 times with 10 volumes of Wash Buffer and elute 4 times with 0.5 volumes of Elution Buffer. Immediately dialyze against several changes of Dialysis Buffer and freeze in liquid nitrogen.

NOTE: The minimum myc epitope tag which is often used in conjunction with the His tag is (EQKLISEEDL). If it is used on the amino terminus add an M and if it is used on the carboxy terminus add an N. The monoclonal antibody which recognizes this epitope is called 9E10 and is described in: Evans, G.I., Lewis, G.K., Ramsay, G., Bishop, J.M. (1985). Isolation of a monoclonal antibody for Human c-myc Proto-Oncogene Product. MCB 5:3610-3616.