Bacterial Expression of Motors

Bacterial Expression of Motors


      Expression plasmid (e.g., pET/Ncd in BL21(DE3)pLysS host cells)

      ZB = 10 g NZ-Amine A

      5 g NaCl

      860 mL final volume

      10X M9 Salts = 1 g NH4Cl

      3 g KH2PO4

      6 g Na2HP04

      100 mL final volume

      M9ZB = 860 mL ZB

      100 mL 10X M9 Salts

      40 mL 10% glucose

      1 mL 1 M Magnesium Sulfate

      1 mL 50 mg/mL Ampicillin in DDW

      0.1 mL 50 mg/mL Chloramphenicol in EtOH

      Sterile glycerol (0.5 mL in sterile tube)

      1 M IPTG, dioxane-free in DDW

      200 mM PMSF in EtOH

    Special Equipment

      Refrigerated Incubator Shaker (e.g., New Brunswick Innova 4330)


      1. Inoculate 1 mL of M9ZB with single colony from newly transformed cells or freshly streaked plate from glycerol culture of newly transformed cells.

      2. Grow cells for 1.5 hr at 37°C.

      3. Inoculate 20 mL M9ZB with the 1 mL culture and grow 1.5 hr at 37°C.

      4. Inoculate 4 x 500 mL M9ZB in 2 L culture flasks with 5 mL of the culture and grow overnight (12-14 hr) at 22°C to OD550 = 0.9 to 1.0.

      5. Sterilely withdraw 0.5 mL cells, mix with 0.5 mL sterile glycerol in a sterile tube, freeze at -80°C for future use as stock.

      6. Add IPTG to 0.4 mM to induce protein expression and shake at 22°C for 3.5-6 hr.

      7. Harvest cells by swirling in ice water bath for 5 min, transfering to centrifuge bottles on ice and centrifuging at 6,500 rpm (7,100 x g) in a Sorvall GS-3 rotor.

      8. Resuspend cells in column buffer containing 0.5-1 mM PMSF and centrifuge in 30 mL tared Oak Ridge tubes.

      9. Weigh tubes, should contain 2-5 g cells/tube.

      10. Flash freeze in liquid N2 and store at -80°C until use.


      1. The use of newly transformed cells or a freshly streaked plate from a glycerol culture of newly transformed cells 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 timepoint, then induce by adding IPTG to 0.4 mM and take 1 hr, 2 hr, 4 hr, 6 hr and overnight timepoints. Centrifuge samples after collection for 1-2 min in a microfuge to collect cells and resuspend pellet in 30 microliters 1X SDS loading dye solution. Analyze by SDS-PAGE (5-10 microliters/lane) to determine the optimal time of induction.

      3. To monitor induction of large cultures, withdraw 0.5 mL of cells from each flask just prior to induction and just prior to harvest. Centrifuge samples for 1-2 min in a microfuge and resuspend pellet in 30 microliters 1X SDS loading dye solution. Analyze by SDS-PAGE to determine if the cells are induced.

      4. For biotinylated motors, grow cells in M9ZB media containing 1 µM biotin and add biotin to 2 µM at the time of induction.

      5. Plasmids may be lost from cells during growth or induction. It is therefore necessary to test each batch of induced cells for protein expression.

    SA Endow

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Created 2 March 1999 21:00 GMT

Modified 29 October 2004 16:45 GMT