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Calmodulin Production

I. Growth and Expression
II. Cell Lysis
III. Protein Purification

Growth and Expression

Transform BL21 (DE3) pLysS cells with Calmodulin plasmid (PiCBWR, supplied by E.Thulin). Follow normal procedure for competent cells except: all incubations after the heat shock step should be at 30°C unless otherwise noted.

Plate transformation reaction on LB agar with 34mg/mL chloramphenicol and 100mg/mL ampicillin. Grow overnight at 30°C, or until colonies form.

Inoculate 20 mL of LB broth (with chloramphenicol and ampicillin as above), grow with shaking overnight at 30°C.

Inoculate 100 mL of LB/chloramphenicol/ampicillin broth with 4 mL of overnight culture. Measure absorbance of starting culture at 600nm. Grow/shake at 30°C until absorbance at 600nm doubles.

Inoculate 900 mL of LB/chloramphenicol/ampicillin broth with entire 100mL culture. Add IPTG to final concentration of 0.5mM. Grow/shake at 37°C until growth stops, about 4 hours.

Harvest cells by centrifugation, freeze cell pellet at -80°C until ready to perform lysis procedure.

Cell Lysis

Prepare Lysis Buffer:
2.4M Sucrose
40mM Tris
10mM EDTA pH=8
add PMSF to final concentration of 1mM.

Prepare Enzyme Buffer:
50mM MOPS
100mM KCl
1mM EDTA
1mM DTT

Resuspend cell pellet in about 10 mL Lysis Buffer (it will be a thick, gooey mess). Incubate suspension on ice for 30 minutes with frequent shaking.

Add about 60 mL Enzyme Buffer, incubate on ice with frequent stirring for 10 minutes.

Add 100 µg/mL lysozyme, incubate on ice with frequent stirring for 1 hour.

Add about 5 U DNAse, and MgCl2 to final concentration of 10mM. Stir at room temperature for 20-30 minutes.

Centrifuge at 20,000 rpm for 20 minutes. Collect supernatant and make 1mM PMSF. Store at -80°C until ready to perform purification procedure.


Protein Purification

Wash Buffer A:
50mM Tris
1mM CaCl2
pH 7.5
Wash Buffer B:
50mM Tris
1mM CaCl2
500 mM NaCl
pH 7.5

Elution Buffer C:
50mM Tris
1mM EDTA
pH 7.5

Prepare two 25 mL phenyl sepharose columns (Amersham Biosciences, #17-1082-01).
Equilibrate Column 1 with 100mL of buffer C.
Equilibrate Column 2 with 100mL of buffer A.

Apply filtered lysate (0.22mm) to Column 1 at about 0.8 mL/minute. Collect flow-through as a single fraction.

Wash Column 1 with 100 mL of buffer C. Collect wash as a single fraction.

Adjust the flow-through fraction from Column 1 to 5 mM CaCl2. Filter and apply to Column 2. Collect the flow-through as a single fraction.

Wash Column 2 as follows:
100 mL buffer A
100 mL buffer B
100 mL buffer A.
Collect each wash as a single fraction.

Elute Column 2 with 100 mL of buffer C. Collect 7 mL fractions. The majority of Calmodulin will elute in the first 40 mL. A very small amount of pure protein may elute in the second A wash.

Analyze fractions by SDS-PAGE. Pool Calmodulin containing fractions and store at -80°C.

Note: the molar extinction coefficient of Calmodulin is 3006 M-1cm-1. By measuring the absorbance of the purified protein pool at 276 nm, an accurate determination of protein concentration can be obtained.

Submitted 08/12/03 by Susan Meyn