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EXPRESSION AND PURIFICATION OF PROTEIN AND WESTERN ANALYSIS

 

Once you have subcloned the cDNA of the myosin heavy chain into a expression vector, you can express the protein in bacteria and purify it using molecular tools that have been built into the expression vector. The purified product can be visualized in a polyacrylamide gel.

Expression
  1. Inoculate 2 ml of SOB with a single recombinant colony
  2. Grow overnight at 37oC with moderate shaking.
  3. The next day inoculate 50 ml of SOB with 1 ml of the overnight culture and grow at 37oC shaking vigorously to an OD600 = 0.6.
  4. Add IPTG to a final concentration of 1 mM and grow at 37 oC with shaking until the optimal time point (~3 hours)
  5. Harvest the cells by centrifugation (3000 x g for 10 minutes at 4 oC)
    6.

Purification

Preparation of Cell Lysates
  1. Re-suspend the cells in 10 ml Native binding buffer ( 20mM NaPO4 , 500 mM NaCl, pH 7.8), add lysozyme (10 mg/ml stock) to 100µg /ml and incubate for 15 minutes on ice.
  2. Sonicate with 2-3 10 second bursts at medium intensity while holding in ice.
  3. Complete the lysis by 3 freeze-thaw cycles. Flash freeze in liquid nitrogen or dry ice/ethanol then warm to 37oC in a water bath.
  4. Add RNase and DNase (1mg/ml stock) to a final concentration of 5 µg/ml each, Incubate at 37oC for 15 minutes.
  5. Remove the insoluble debris by centrifugation (3000 x g for 15 minutes).
  6. Clear the lysate by passage through a 0.8 µm syringe filter.
    7.

Preparation of the column
  1. Re-suspend the resin in a column by inverting and gently tapping the column.
  2. Place the column in a 15 ml tube and centrifuge at 800 x g in a swinging bucket rotor for 2 min to pack the resin.
  3. Carefully remove the column from the centrifuge. Take the closure off and gently aspirate the buffer, being careful not to remove any of the resin. Add 7 ml of sterile distilled water and replace the top closure. Re-suspend the resin as in Step 1 by alternately inverting and gently tapping the column.
  4. Centrifuge as above, then gently remove the water. Repeat this water wash one additional time.
  5. Add 7 ml of Native Binding Buffer, pH 7.8. Resuspend the resin, centrifuge and aspirate as before. Perform this step three times.

Protein Binding
  1. Batch bind the protein by re-suspending the equilibrated column with a 5-ml lysate aliquot. Gently rock the column for 10 minutes to keep the resin suspended and to allow the polyhistidine-tagged protein to fully bind. Settle the resin by low speed centrifugation (800 x g) and carefully aspirate the supernatant. Repeat with a second 5 ml aliquot.

Column Washing and Elution of the Protein
  1. Wash the column 3x with 4 ml of Native Binding Buffer, 7.8 by re-suspending the resin, rocking for 2 min and then separating the resin from the wash solution by centrifugation.
  2. Wash the column 5 x as above with 4 ml Native Binding Buffer, 6.0
  3. Wash the column 2x with 4 ml of Native Wash Buffer, 5.5.
  4. Clamp the column in a vertical position and snap off the cap on the lower end. Elute the protein by applying 5 ml of Native pH Elution Buffer. Collect 1 ml fractions. Monitor the elution by taking OD280 reading from the fraction.
  5. Check the size and estimate the concentration of the purified product by running a sample in a polyacrylamide gel.