Protocol 7 Purification of Histidine-tagged Proteins by Immobilized Ni2*+ Absorption Chromatography 15.44 igificant affinities to such matrices, His--labeled proteins gen erated by recombinant techniques can be purified substantially in a single step by metal chelate atfinity chromatography. Those natural proteins that do bind to these matrices ean almost alivays be removed in a second chromatography step. Metal chelate chromatography has become popw lar because it is both highly effective and relatively insensitive to proper protein folding, ionic strength, chaotropes, and detergents (Hochuli etal. 1988). Because of the high efficieney high capacity concentrating power, and speed of this technique, it should be used asa first (and some: times only) step in a purification protocol. To perform metal chelate chromatography, the protein of interest must first be engineered to contain a His-6 sequence in an exposed and relatively flexible site, frequently found at the amino or carboxy terminus (Van Reeth et al, 1998). It can be helpful to place one or two glycine residues between the His-6 sequence and the rest of the protein. Unique protease cleavage sites also can be inserted to allow removal of the His-6 sequence after purification (please see Table 15-3), Proteases may also be used to desorb proteins from the affinity matrix by cleavage at the linket/His-6 insertion. Coexpression of an unmodified protein of interest together with a His-6 tagged binding partner can allow isolation ofthe binary complex on the affinity matrix and selee tive elution of the native target protein (Kozasa and Gilman 1995), This method can be partiew larly effective if the two proteins can be dissociated gently such that contaminating proteins that bind to the matrix are not eluted (please see Chapter 18, Protocols 2 and 3). Purification can be performed under both native and denaturing conditions (eg. in buffers containing § M urea or 6 M guanidine hydrochloride}. Because any chelator can interfere with metal chelate chromatography, buffers used for extraction and chromatography should be free of EDTA or EGTA, Submillimolar concentrations of dithiothreitol may not interfere signiticantly. Nevertheless, it is preferable to use butfers containing 1-5 mt of a monothiol such as f-mercap toethanol or B-mercaptoacetic acid. The target protein is eluted by a chelator such as imidazole or EDTA. Imidazole is the more selective eluant and should be used whenever possible, Efficient clution is usually achieved with 50-100 mM imidazole at pH 7-8. The lowest effective concentraProtocol 7: Purification of Histidine-tagged Proteins by Immobilized Ni Absorption Chromatography 15.45 tion should be established using a linear gradient of 10-150 mM imidazole. EDTA is more effec- tive, but it may strip Ni?* from the NTA-agarose. In this case, the Ni°* must then be removed for ‘many applications, either by dialysis or by a second chromatographic step. ‘The Né* resin can be easily regenerated and reused many times (please see the additional protocol at the end of this protocol). Because of its large binding capacity, as much as 20 mg of recombinant protein may be purified on a 2.5-ml column. The following protocol is adapted from ‘methods contributed by Elliot Ross (University of Texas Southwestern Medical Center, Dallas) and Tzu-Ching Meng in Nick Tonks’ group at Cold Spring Harbor Laboratory MATERIALS CAUTION: Please see Appendix 12 for appropriate han Buffers and Solutions Please see Append Dilute stock sol Binding butfer (pH 7.8) 20 mt sodium phosphate 500 mM NaCl Imidazole elution butter (pH 6.0) 20 mst sodium phosphate 500 mM NaCl Createa series of four elation buffers containing imidazole at concentrations of 10 ms, 50 my, 100 mi and 150 mM by adding the appropriate amount of 5X1 imidazole to wash baer (pH 6.0), Titon X-100 (10% viv) Wash buffer (pH 6.0) 20 mst sodium phosphate 500m NaC 1 for components af stock solutions, buffers, and reagents. ns to the appropriate concentrations. Enzymes and Buffers DNase (1 mgiml) lysozyme RNase (1 mgiml) Gels Polyacrylamide gel (10%) containing SDS For preparation of SDS-polyacrylamide gels used inthe separation of proteins, please see Appendix Centrifuges and Rotors Sonall $5-34 rotor or equivalent Special Equipment Chromatography column (glass or polypropylene)15.46 Chapter 15: Expression of Cloned Genes in Escherichia coli Niz~-charged chromatography resin (e.g., ProBond, Invitrogen; HisTrap, Pharmacia; HissBind Resin, Novagen; NTA-Ni"-agarose, Qiagen) Gurreaty, the most common metal chelate affinity support is nitrilotriacetate (NTA Ni? -agaros, available from the suppliers described above, Other mattices with different propertis are aso wailaie A proprietary Coe chelate (Talon) ssid to bind His-tagged proteins wth higher affinity, and some ‘what higher selectivity, bu it cannot be conveniently regenerate in the laboratory and is consequently much mote expensive Rocking platiorm Vectors and Bacterial Strains E.coli cells expressing a polyhistidine-ta ed protein (generated in Protocol 1, 2, 3, or 4) METHOD Preparation of the Ni?* Affinity Column 1. Gently invert the bottle of Ni?*-charged chromatography resin to mix the slurry, and trans- fer 2 ml toa small polypropylene or glass column. Allow the resin to pack under gravity flow. Wash the resin with 3 cohumn volumes of sterile HO. (One cokimn volume is equialent othe vlome of the sete bed of rein Equilibrate the resin with 3 column volumes of binding buffer (pH 7.8). The column is now ready for use in Step 9 Preparation of Cell Extract 4. Resuspend the cell pellet (e.g. from Protocol 1, Step 17) in 4 mil of binding butfer (pH 7.8) per 100 ml of cel culture Add lysozyme toa final concentration of I mg/ml and incubate the cell suspension on ice for 30 minutes. Several protocols use sonication or a French press to lyse ells before affinity purification of the polphisidine-tageed protein, The use of lysozyme, a8 described her, is somewhat more repr - Additional Materials Low pH elution buffer (pH 4.0) 20 m sodium phosphate 500 mat Nact Use phosphoric acid to lower the pH 0 40. Wash bute pH 5.5) "20 mat sodium phosphate 500 mat NaCl Method 1. Carryout the main protocol through Step 12 2. Wash the column with 6 volumes of Wash buffer tl pH 5.5) | 3. Elute the bound protein wth 6 volumes of low pH elution buffer (pH 4.0), Collect t-m rations from the column, and monitor the Argo ofeach faction, 4. Assay the fractions of interest for the presence of the polhistdinetagged protein by analyzing 20+! aliquots by electrophoresis through a 10% SDS-polyaerylamide gel ‘|15.48 Chapter 15: Expression of Cloned Genes in Escherichia coli ADDITIONAL PROTOCOL: REGENERATION OF NTA-NI?*-AGAROSE INIANN2*-agarose canbe reused almost indefinitely if its washed well and regenerated. the column has not | been oxidized or depleted ons fight blue color or appearance of yllow/brown colo, the manulacturee ec commends washing with 0.2 M acetic acid in 30% glycerol. We routinely stip and regenerate the matrix after | every use according to the manufacturers recommendation (Qiagen inthis cas). CAUTION: Please see Appendix 12 for appropriate handling of materials marked with . Adina Maeals | Sigs code 2 catia <> Sh ioss 2s 3m and 75 ta a) roo mur co1A rans, Method 1. Wash the colin successively with | 2 volumes of 6 Mi guanidinium chloride'0.2 M acetic acd (or other stripping agent) 5 volumes of HO | | 2 volumes of 2% SDS 1 volume each of 25%, 50%, and 75% ethanol resale | Nekiecthe 8h sox ana a eres | | 5 volumes of 100 m\4 EDTA habeas |