Growth, Purification, and Refolding of&n

Description
Growth, Purification, and Refolding of TGF-b3 (for 3L)

Procedure
1) Transform BL21(DE3)cells with pET32 plasmid containing the TGF-b3 sequence

a) Place 10ml competent cells in a microfuge tube and add ~0.5mg plasmid DNAon ice.

b) Incubate on ice 30 min.

c) Incubate @ 42¡ãC for 45 sec.

d) Immediately place on ice for 2 min.

e) Add 200ml LB medium.

f) Incubate @ 37¡ãC with shaking (250 RPM) for 30 minutes.

g) Plate 75 ml on LB plate containing 100 mg/ml ampicillin.

h) Culture for 10 hours or until colonies can be seen.

2) Culture cells

a) Inoculate 3 x 1L LB medium (or M9 when isotopically labeling) pH 7.4 containing 200 mg/ml ampicillin with single coloniesfrom the plate.

b) Culture @ 37¡ãC with shaking (250 RPM) until the O.D.600 is0.6-1.0. (~6 hrs.)

c) Induce protein expression by adding 100mg IPTG.

d) Continue culture for 3 hours.

e) Harvest the cells by centrifugation in 1L bottles @ 9000 x g for 7 min.

3) Recover non-soluble,monomeric TGF-b3 (all stepsat 4¡ãC)

a) Resuspend cell pellet in 60 ml 100mM Tris/HCl, 10mM EDTA, 1mM PMSF, pH @4¡ãC = 8.3 (disruption buffer)(make stock and add PMSF dissolved in isopropanol just prior to use).

b) Pass cell suspension through French Pressure Cell twice.

c) Add disruption buffer to a total volume of 200ml.

d) Centrifuge 20 min. @ 15,000 x g.

e) Resuspend pellet in 100 ml disruption buffer containing 1M NaCl.

f) Centrifuge 10 min. @ 15,000 x g.

g) Resuspend pellet in 100 ml disruption buffer containing 1% Triton X-100 (v/v).

h) Centrifuge 10 min. @ 15,000 x g.

i) Resuspend pellet in 50 ml 20mM Tris/HCl, 1mM EDTA, 1mM PMSF, 1%DTT (w/v),pH @ 4¡ãC = 8.3. (Add PMSF and DTT prior to use)

j) Homogenize in a tissue grinder (5-10 strokes).

k) Add glacial HAc to 300 mL total volume (pH should be near 2.5).

l) Stir for 1-2 hours as inclusion bodies take some time to dissolve

m) Centrifuge10 min. @ 15,000 x g.

4) Initial purification.

a) Equilibrate a 2.5 x 70 cm Sephacryl S-100 column overnight with 10% HAc.

b) Concentrate supernatant above to about 7 ml using Amicon stirred cell withYM3 membrane.

c) Load onto the column and collect about 7ml fractions (use the ISCO systemusing either gravity or the pump (set at X1, and 90) at a column flow rateof ~ 1 mL/min; set chart recorder to 2.o Absorbance Units Full Scale (AUFS),chart speed to3 cm/hr, and program the fraction collector to collect fractions6-8 min per tube).

d) Pool the fractions containing the TGF-b3 (Second peak; generally elutes between190 and 220 mL)

e) Concentrate the fractions to 10 ml and assay concentration by A280 absorbance (conc. ¡Ö 0.61mg/ml per O.D. unit) (make dilutions and spectrophotomerblank in 100 mM HAc)

5) Refolding (13¡ãC)

a) Add concentrated fractions dropwise to enough 100mM Tris, 30mM CHAPS, 1MNaCl, 5mM reduced glutathione and 20% (v/v) DMSO pH at 13¡ãC = 9.5 (refoldingbuffer) while stirring gently to have a final TGF-b3 concentration of 0.2mg/ml (higher concentrations of protein and lower CHAPS concentrations mightbe O.K., although this hasn¡¯t yet been thoroughly checked). The pH should be monitored during addition so that its keptbetween 9.65 and 9.75. Once the addition is complete lower the pH to 9.50.
WEAR GLOVES!

b) Continue stirring for 24-48 hours uncovered at 13¡ãC (monitor the bathvolume--if it gets too low, the temperature increases)

c) Acidify the solution by adding glacial HAc to a pH of 3.5.

d) Dilute the solution approximately 1.5 fold using H2O (readjustthe pH back to 3.5 with Hac as necessary).

e) Concentrate to ~ 50 ml @ 4¡ãC in a 400 ml Amicon stirred cell using aYM3 membrane.

f) Dialyze 3X versus 4L 100mM HAc @4¡ãC

g) Centrifuge 15 min. at 6,000 x g in a 50 ml conical centrifuge tube.

h) Decant supernatant and assay concentration by A280 absorbance(save pellet for possible recovery)

6) Isolation of dimer

a) Equilibrate a Source 15S HR 10/10 column with 20mM NaAc, 30 % isopropyl alcohol,pH 4.0 (Buffer A) at a flow rate of 2.0 ml/min.

b) Load ¡Ü 50mg and wash column with 1.5 Column Volumes Buffer A.

c) Elute with a linear gradient over 10 column volumes from 0% to 40% BufferB (Buffer A + 1M NaCl).

d) Collect peak fractions.

e) Assay concentration by A280 absorbance.

7) Final purification

a) Load ¡Ü 1mg onto Vydac 214TP54 column pre-equilibrated at a flow rate of 1ml/min. with 75% 0.1% TFA in H2O. (Solvent A) / 25% 0.1% TFA inMeCN (Solvent B).

b) Wash with5 Column Volumes 25% Solvent B.

c) Elute with linear gradient from 25% to 45% Solvent B over 8 Column Volumes.

d) Pool collected fractions and lyophilize aliquots in eppendorf tubes for storageat -80¡ãC.

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Growth, Purification, and Refolding of&n