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Expression

Grow cells at 30°C.

Innoculate 1L of LB or TB + Amp with 10mL of overnight culture.

Induce cells with 0.5mM IPTG at OD ~0.7

After ~ 3 hrs harvest cells by centrifugation, 20m x 4krpm

Freeze cells (-80°C) with 5mL H6 protein lysis buffer.

Purification

Thaw with additional 5mL lysis buffer and protease inhibitor (Roche EDTA free protease inhibitor capsules work well)

Lyse by your favorite method

Centrifuge- 30m x 15krpm

Ni-NTA column pre-incubate supernatant with washed Ni2+-NTA slurry. (~30m with shaking in cold room)

Wash with ~50mL NB

Elute with ~10mL NE, take 1mL fractions.

Concentrate as needed.

For all buffers see Sauer:Purification_of_His-tagged_proteins/Native_prep

Concentration can be determined with extinction of 19 770cm-1 at 280nm