1 Purification and Characterization of T7 RNA Polymerase Experiment 1 of 2 CHEM 4581: Biochemistry Laboratory I Version: February 16, 2008 Goal. The gene for a protein believed to be His-tagged T7 RNA polymerase has been overexpressed in E. coli cells. The goal of this study is to isolate, purify and characterize the His-tagged protein that has been over-expressed in order to confirm that it is T7 RNA polymerase. Experimental Design Strategy. Many analytical methods can be employed to confirm the identity of the overexpressed protein. The purification and characterization of T7 RNA Polymerase to be done here has 5 parts: (1) Cell lysis (2) His-tag affinity chromatography (3) Buffer exchange via ammonium sulfate precipitation (4) Protein concentration determination via the Bradford Protein Assay (5) MW determination via SDS-PAGE. Students will be provided with a frozen pellet of E. coli cells corresponding to 100-200 mL of bacterial culture. The cells must first be lysed open and the protein separated from other biological macromolecules, cellular organelles, membrane components and any other cellular components. Cell lysis can be performed using chemicals (such as NaOH), enzymes (such as lysozyme), or some form of physical agitation or disruption (such as homogenization or sonication). Here, students will use ultrasonication to lyse open the bacterial cell pellets after resuspension. Proteins can be artificially tagged with histidine residues at either the N- or C-terminus to deliberately exploit metal affinity chromatography as a purification strategy. The purification can be so efficient, due to the high specificity of ligand binding to the resin, that only one-step of purification is needed oftentimes. Here, students will use Novagen’s His-Bind® resin charged with Ni2+ to purify any His-tagged protein in their bacterial cells using affinity chromatography. Bound protein must be eluted with a molecule that will compete with the His-tag for binding to the Ni2+ on the resin. A reasonable competitor molecule would be one that has a similar structure to histidine, but with more degrees of freedom for movement and at substantially higher concentration than the His-tagged protein. The conventional reagent used to elute His-tagged proteins is imidazole at concentrations on the order of 200 mM. Protein eluted from a metal affinity chromatography column would typically be captured in buffer containing a relatively high concentration of imidazole, which interferes with many subsequent methods such as colorimetric protein assays, gel electrophoresis, and some activity assays. Therefore, a buffer exchange step is required prior to analytical testing or storage of the protein until later analysis. There are many strategies for conducting buffer exchange including dialysis, immunoprecipitation, and filtration. One economical approach is simply ammonium sulfate precipitation followed by resolubilization of the precipitated purified protein in an imidazole-free buffer. Students will end the day’s work by performing ammonium sulfate precipitation on their samples prior to storage at 2-8°C.2 MATERIALS AND REAGENTS Bacterial Overexpression  LB Broth  LB Agar  10 mg/mL Ampicillin  IPTG  Inoculating Loop  Flame  Bacteria  Autoclave Metal Affinity Chromatography  100 mM Phenylmethanesulfonyl fluoride (PMSF) - a protease inhibitor  100% (w/v) Sodium Deoxycholate - a detergent  Sorvall RC5 Super-speed ultracentrifuge  FiberLite® F13-50cy rotor that accepts 50-mL conical tubes (Piramoon Technologies, Inc.)  Branson Sonicator Model 250A  Shimadzu UV1601 UV-visible spectrophotometer  Novagen His-Bind® Resin  Bio-Rad Glass Econo-Column (ID: 1.0 cm; Length: 10 cm; Max Vol.: 8 mL)  Tygon Tubing, Formulation R-3603  50 mM Nickel Sulfate  Ammonium Sulfate Binding Buffer  20 mM Tris-HCl, pH 7.9  0.5 M NaCl  0.05% (v/v) Tween 20 (detergent)  5 mM Imidazole Wash Buffer  20 mM Tris-HCl, pH 7.9  0.5 M NaCl  0.05% (v/v) Tween 20 (detergent)  60 mM Imidazole Elution Buffer  20 mM Tris-HCl, pH 7.9  0.5 M NaCl  0.05% (v/v) Tween 20 (detergent)  200 mM Imidazole Strip Buffer  0.2 M Tris-HCl, pH 7.9  0.5 M NaCl  0.1 M EDTA3 EXPERIMENTAL PROCEDRES Performed by the TA - Growth of E. coli Cells Overexpressing T7 RNA Polymerase Day 1:  Prepare the agar solution according to the manufacturer's instructions.  Autoclave the agar solution then allow it to cool to ~50 ºC.  To the agar solution, add 1 μL of 100 mg/mL ampicillin for every μL of agar. Pour the agar onto a petri dish and allow the agar to solidify.  Streak the plate with the IPTG-inducible recombinant expression vector containing His-tagged T7 RNA polymerase using a sterile inoculating loop. Cover the petri dish with its top and wrap parafilm around the circumference.  Leave the plate inverted at 37 ºC for ~16 hours (overnight). Day 2:  Check the agar plate for bacterial growth. Store the plate at 2-8 ºC until further use.  Make LB broth according to the manufacturer's instructions. Autoclave the broth in ~500 mL aliquots in 2L Erlenmeyer flasks. Keep the flasks covered with foil to deter contamination. Also make a flask containing 100 mL of LB broth in 500 mL flask to generate a starter culture. Allow the broth to cool.  Add enough ampicillin to the starter culture for a 60 μg/mL solution. Allow the solution to mix for 10 minutes.  Inoculate the starter culture with a single colony from the agar plate.  Leave the starter culture at 37 ºC for 16 hours with vigorous shaking (i.e. setting #4 on shaking incubator). Day 3:  Add enough ampicillin to the large cultures for a 60 μg/mL solution per flask. Allow the solution to mix for 10 minutes.  Inoculate the large cultures with 1 mL of the starter culture per 100 mL of culture.  Allow the larger cultures to shake vigorously at 37 ºC until the absorbance of the culture reaches ~0.6 at 600 nm. (This normally takes ~2-2.5 hours).  Add IPTG to a concentration of 0.4 mM to induce bacteria to produce T7 RNA polymerase. Do this by preparing a concentrated solution and adding a very small volume to the culture. The subsequent vigorous shaking will allow for thorough mixing.  Incubate at 37 ºC with vigorous shaking for 4 more hours.  Distribute the culture into 200 mL aliquots in Sepcor bottles.