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MOLECULAR BIOLOGY: WORKING WITH DNA

EXPRESSION: FUSION PROTEINS

Induction of Bacterial Expression of Heterologous Motor Proteins

Induction of Bacterial Expression of Heterologous Motor Proteins
 
Overview
This protocol describes the process of induction of bacterially expressed fusion proteins. In particular, this protocol describes the induction process for the expression of motor proteins fused to a heterologous promoter.
 
Procedure
1. Inoculate 1 ml of M9ZB Media with a single colony from bacterial cells newly transformed with plasmid containing gene encoding protein of interest or from a plate freshly streaked from a glycerol culture of newly transformed cells (See Hint #1).

2. Grow the cells for 1.5 hr at 37°C with shaking.

3. Inoculate 20 ml of M9ZB Media with the 1 ml culture.

4. Grow cells for 1.5 hr at 37°C.

5. Inoculate four 2-liter culture flasks containing 500 ml of M9ZB Media with 5 ml of the culture.

6. Grow cells overnight (12 to 14 hr) at 22°C until the Optical Density at 550 nm is between 0.9 and 1.0. (OD550 = 0.9 to 1.0).

7. Withdraw 0.5 ml of the cells and mix with 0.5 ml Sterile 50% Glycerol in a sterile tube.

8. Freeze the glycerol-cell suspension at -80°C for future use as a stock culture.

9. Add IPTG to the Culture Media to a final concentration of 0.4 mM to induce protein expression and shake at 22°C for 3.5 to 6 hr (See Hint #2).

10. Harvest the cells by swirling in an ice water bath for 5 min.

11. Transfer the cells to centrifuge bottles on ice.

12. Centrifuge at 6,500 rpm using a Sorvall GS-3 rotor (7,100 X g) at 4°C for 10 min.

13. Discard the supernatant.

14. Resuspend the cells in appropriate buffer containing 0.5 to 1.0 mM PMSF.

15. Flash freeze the cells in Liquid Nitrogen and store at -80°C until needed or continue by lysing cells and purifying protein of interest.

Solutions
1 M MgSO4   Autoclave
10% (w/v) Glucose   Autoclave
M9 Salts (10X)   30 mg/ml Potassium Phosphate Monobasic (KH2PO4)
10 mg/ml NH4Cl
Autoclave
60 mg/ml Sodium Phosphate Dibasic (Na2HPO4)
ZB Solution   11.63 mg/ml NZ-Amine A
Autoclave
5.81 mg/ml NaCl
200 mM PMSF   (CAUTION! see Hint #2)
Prepare in 100% Ethanol
200 mM Phenylmethylsulfonyl Fluoride (PMSF)
1 M IPTG   1 M Isopropyl β-D-Thiogalactopyranoside (IPTG), dioxane-free
Sterile 50% Glycerol   50% (v/v) Glycerol
Filter sterilize
M9ZB Media   860 ml of ZB Solution
1 ml of Ampicillin Stock
1 ml of 1 M MgSO4
100 μl of Chloramphenicol Stock
100 ml of 10X M9 Salts
40 ml of 10% (w/v) Glucose
Chloramphenicol Stock   Prepare in 100% Ethanol
50 mg/ml Chloramphenicol
Ampicillin Stock   50 mg/ml Ampicillin
Filter-sterilize
 
BioReagents and Chemicals
Sodium Chloride
Magnesium Sulfate
Chloramphenicol
Isopropyl beta-D-Thiogalactopyranoside (IPTG)
NZ-Amine A
Glucose
Ampicillin
Ethanol
Phenylmethylsulfonyl Fluoride (PMSF)
Glycerol
Potassium Phosphate, Monobasic
Sodium Phosphate, Dibasic
Ammonium Chloride
 
Protocol Hints
1. The use of newly transformed cells or a freshly streaked plate from a Glycerol culture of newly transformed cells greatly improves protein expression.

2. To optimize induction time, grow a 20 ml culture to OD550 = 0.9 to 1.0, withdraw 0.5 ml for 0 time point, then induce by adding IPTG to 0.4 mM and take 0.5 ml aliquots at 1 hr, 2 hr, 4 hr, 6 hr and overnight time points. Centrifuge the samples after collection for 1 to 2 min in a microcentrifuge to collect the cells. Resuspend the pellet in 30 μl of 1X SDS loading dye solution. Analyze by SDS-PAGE (5 to 10 μl/lane) to determine the optimal time of induction.