Experiment 2.3 Purification of T7 RNA Polymerase
(version: 01/12/00)

Introduction

The target protein in this laboratory is T7 RNA Polymerase (molecular weight of 99,000 Daltons). This protein makes RNA transcripts from DNA templates, and will be used in subsequent laboratory exercises to synthesize RNA. The TA will grow a 250 ml prep of E. coli that overproduces his-tagged T7 RNA polymerase, will spin the culture down in a centrifuge, and give you the pellet. After purifying the target protein from the pellet, students will run an SDS-PAGE gel to verify that your protein is pure.

Supplementary Reading Material

Voet & Voet: p.p. 919-930 (RNA polymerase); p.p. 71-101 (Protein Purification esp. the section on Affinity Chromatography)

Journal Article: Durbin et al, Expression and Purification 9, 1997, 142-151.

A. PREPARATION OF INDUCED BACTERIA

LB Broth
NaOH to pH LB Broth
IPTG
Inoculating Loop
Flame
Bacteria
Autoclave

B. HIS TAG COLUMN CHROMATOGRAPHY

Stock Solutions

Component	Stock Concentration	Volume/500 mL
200 mM Tris-HCl, pH 7.9	1 M	10 mL
500 mM NaCl	5 M	50 mL
0.05% Tween-20	10% (v/v)	2.5 mL

 

Binding Buffer	above with 5 mM imidazole	1.702 g
Wash Buffer 1	above with 20 mM imidazole	2.723 g
Wash Buffer 2	above with 60 mM imidazole	8.169 g
Elution Buffer	above with 200 mM imidazole	68.080g

Charge Buffer: 50 mM NiSO4 in ddH2O

Storage Buffer: 20 mM Na2HPO4/NaH2PO4, pH 7.4, 1 mM EDTA, 100 mM NaCl, 50 % glycerol (v/v)

Strip Buffer: 200 mM Tris, pH 7.9, 500 mM NaCl 100 mM EDTA

D. ELECTROPHORESIS OF T7 RNAP

High Molecular Weight Markers

A. GROWTH OF BACTERIA (performed by TA)

Make LB broth containing 60 microgram/mL ampicillin

Inoculate 100 mL of LB with single colony and grow overnight

Next day inoculate 0.1 liter with 1 mL of the overnight stock

When OD gets to ~0.6 at 600 nm add IPTG to a concentration of 0.4 mM to induce bacteria to produce protein.

Incubate for 4 more hours

Centrifuge at 12K RPM for 15 minutes

Discard supernatant

Freeze pellet at -20 Celsius

B. CELL LYSIS (performed by students)

(Perform B and C simultaneously)

Thaw the pellet on ice

Dissolve the pellet in 10 mL binding buffer and vortex

Add 12 microliters 100 mM PMSF (protease inhibitor) in ethanol and 100 microliters 10% sodium deoxycholate (detergent), and vortex

Incubate on ice for 10 minutes

Sonicate five times for 20 seconds each to break open cells (keep in ice)

Centrifuge at 15K for 15 minutes at 4 degress C in the special high speed tubes given to you by your TA.

C. His-Tag Column Purification

A 1 mL column will have been prepared by the TA. A 50% ethanol/nickel agarose resin slurry was added to the column. Allow the column to drain. Wash column with 5 mL dd H2O. Equilibrate the column with 5 mL binding buffer. Add 5 mL of "charge buffer" to charge the column with nickel. Rinse out excess charge buffer with 5 mL binding buffer. Filter your protein sample with a 0.45 micron syringe filter to remove particulates.

Load the sample onto the charged column.
Wash with 5 mL binding buffer.
Add 5 mL wash buffer 1
Add 1 mL wash buffer 2
Allow column to stop dripping
Add 4 mL elution buffer and collect two 2 mL fractions
Remove 10 microliters of sample to run on SDS-PAGE gel.
Strip the nickel from the column using 5 mL strip buffer.

Reseal the column with caps as soon as liquid reaches top of column. The column should not be allowed to drain completely. DO NOT DISCARD COLUMN OR RESIN!

D. Quantification/Concentration/Storage

Take OD (determine the absorbance) in quartz cuvettes at 280 nm using 0.2 mL of fraction and 0.8 mL elution buffer.
Calculate yield using the following formula - mg. protein = (99*5*5*OD)/1.4x10^5

Use Macrosep 50K cutoff filter and centrifuge at 6K RPM at 4°C for 30 min.

Fill Macrosep with storage buffer and centrifuge for at least 30 minutes. Repeat at least three times

Dilute sample to a final concentration of 1 mg/mL.

Aliquot into 100 microliters fractions and store in 600 microliter Eppendorf tubes.

Freeze at -20 degrees C

Important Note: The lifetime of your enzyme is dependent on near total replacement of elution buffer with storage buffer. This step can make the difference between the enzyme lasting a week or for months. If a solution freezes at -20 then it contains too much elution buffer.

E. SDS PAGE

Prepare and run an SDS PAGE gel as described under Analytical Procedures on the syllabus page. Use high molecular weight markers since T7 RNA polymerase is 99,000 Daltons.

The molecular weight of these markers are:

protein	Molecular weight (Daltons)
Myosin	205,000
Beta-galactosidase	118,000
BSA	85,000
Ovalbumin	47,000

1) What is the purpose of the Sodium Deoxycholate and PMSF in the isolation protocol?

2) Explain the formula given in the procedure for calculating protein mass.

3) Calculate your yield (mg protein/L of LB) for T7 RNA polymerase.

4) Explain BRIEFLY how acrylamide gel electrophoresis works. How does the acrylamide concentration effect the mobility? Explain chemically how the gel polymerizes.

5) Calculate the relative mobilities of all major bands on the gel. If you cannot see the dye front, use the end of the gel as a guide.

(Relative mobility = distance moved by protein/distance moved by dye)

6) Plot a graph of mobility versus log molecular weight of the molecular markers.

HINT:

It can be difficult to determine the identity of the molecular weight markers. Keep in mind that the largest molecular weight marker will be near the top of the gel and the furthest away from the dye front, and will definitely be visible. Some of the smaller molecular weight bands may wash out of the gel, so you may not be able to see all of the marker proteins.

7) Determine the molecular weights of each band on the gel. Prepare a table that lists the band number, the relative mobility, and molecular weight of all the major bands in the gel.

8) Has your purification worked? How might you further purify the product?

9) Suppose you have Protein X. Your goal is to mass produce it in the same manner as that of the T7 RNAP you just purified. Draw a schematic diagram of how would you go about getting a bacteria to overproduce your protein? (Hint: See the assigned article for help).

10) Use Rasmol to measure distances, and to construct a table of ten amino acids of T7 RNA polymerase that are within 6.0 Å of the nucleic acid to which it binds. The table should have five columns (1. protein residue name and number, 2. protein atom name and number, 3. nucleic acid residue name and number, 4. nucleic acid atom name and number, and 5. distance)