Purification of dnEBNA-1/Soft from E. coli BL21 LysS

Inoculate 2ml of 5ml o/n culture of either p3133 (empty vector pET11a)or p3134 (dnEBNA-1/Soft)in E.coli BL21 LysS per 0.5L LB ampicillin (grow two 0.5L cultures of each)

Incubate ~2hrs 37℃250rpm until OD₆₀₀ = 0.4-0.6

Induce with 5ml 100mM ITPG per 0.5L culture

Incubate 2-3hrs 37℃250rpm

Spin down 2 flasks of each (1L total)in 500ml GSA centrifuge bottles 5000rpm,4℃ 10min

Decant supernatant,freeze pellet @20℃

Resuspend pellet in TED 0.15M NaCl (2-5ml per gram wet weight of pellet; for bacterial pellet from 1L I used 6ml on 02/2004)

Add 200x lysozyme and 100x proteasome inhibitors to 1x final concentration

Transfer to disposable,sterile tube and incubate on ice 30min (omitted on 02/2004)

Sonicate setting 10,15-30sec bursts,6 rounds,incubate on ice ~2min between rounds

Transfer to microfuge tubes; Centrifuge4℃ 10k rpm 30min

Transfer supernatant to fresh tube.

Column Preparation

a.Resuspend matrix as 50% slurry and load closed column

b.Wash twice with 5 bed volumes of TED 0.15M NaCl

c.Open column and drain to form compact 100% slurry

Load sample onto column.Note: all flow rates should be 0.5ml/min

Collect Flow Through (FT),pass FT over column again.Save 50μl of supernatant as FT.

Wash with 25ml TE 0.15M NaCl.Collect this wash and save 50μl as Wash 1.

Wash with 15ml TE 0.5M NaCl,collect in 5ml fractions.Save 50μl of each as Wash 2.1,Wash 2.2,Wash 2.3.

Elute dnEBNA-1/Soft from column with 5-10ml TE 0.7M NaCl30% propylene glycol,collect in 1ml fractions.Add 5μl of 0.02M DTT to each 1ml fraction.Save 50μl of each fraction as Elution 1,2,3,4,5 (6,7,8,9,10).

Run 2 identical SDS-PAGE gels with all bold samples above,protein size marker,and concentration standards (BSA 0.1μg,1μg,10μg)or protein positive for Soft-tag and/or EBNA-1

d.Run one gel as a Coomassie Blue stain for bulk protein levels

e.Run one gel as Western for identification of Soft tag and/or EBNA-1

TED 0.15M NaCl

10mM Tris pH 7.4

1mM EDTA (pH 8)

150mM NaCl

0.1mM DTT

TE 0.5M NaCl

10mM Tris pH 7.4

1mM EDTA (pH 8)

500mM NaCl

10% glycerol (v/v)

TE 0.7M NaCl30% propylene glycol

10mM Tris pH 7.4

1mM EDTA (pH 8)

750mM NaCl

30% propylene glycol (v/v)