Preparation and Refolding of Tailspike Inclusion Bodies

SB 8/10/99

Preparation of tailspike inclusion bodies from 1 Liter E. coli culture

1. Resuspend pellet from lysozyme-treated & DNA-treated lysate in 50-ml buffer B (pH 7.6), 0.2 % octylglucoside.

2. Incubate 1 hour on ice with occasional mixing.

3. Pellet IBs at 10K fo 10 min in GSA rotor (4°C). Pellets from induced cells should pack nicely, & pellets from uninduced cells should be loose.

4. Resuspend in 50-ml buffer B. Store aliquots at -20°C. Determine concentration of tailspike chains by SDS-PAGE & densitometry.

Refolding detergent-washed tailspike inclusion bodies

1. Pellet inclusion body sample containing 50 µg tailspike chains (2 min at 14K & 4°C in microfuge). Aspirate supernatant.

2. Dissolve pellet in 6 M urea, 10 mM DTT, 50 mM Tris-HCl (7.6), 25 mM NaCl, 2 mM EDTA. Pellet dissolution may require pumping several times with P200.

3. Incubate for 1 hour at RT. Vortex occasionally.

4. Remove insoluble material (5 min at 14K & 4°C).

5. Chill supernatant on ice & dilute 10-fold with ice-cold buffer B.

6. Shift to 20°C after 1 hour on ice.