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Hinck Laboratory - Lab Protocols
Growth, Purification, and Refolding of N-terminal HisTagged TGFb3
Cell Culture
a) Transform the His6-TGFb3 construct in plasmid pET32a (VOBASE ID 88) into Stratagene BL21(DE3) cells and plate onto LB/AMP. Grow O/N.
b) Pick 3 colonies with a toothpick and toss into 1.0L of LB medium with 150 ug/mL AMP.
c) Induce protein expression at A600 = 0.6 with 230 mg IPTG. Grow cells an additional 6 hr. Collect cells by centrifugation and freeze pellets at 20 C.
Inclusion Body Isolation (volumes indicated are per liter of cultured cells)
a) Resuspend cell pellet in 45 ml 100mM Tris/HCl, 10mM EDTA, 1mM PMSF, pH @4C = 8.3 (disruption buffer)(make stock and a
dd 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 70 ml.
d) Centrifuge 20 min @ 15,000 x g.
e) Resuspend pellet in 100 ml disruption buffer containing 1M NaCl (use the tissue grinder to make even suspension if difficult otherwise).
f) Centrifuge 10 min @ 15,000 x g.
g) Resuspend pellet in 100 ml disruption buffer containing 1% Triton X-100 (use the tissue grinder to make even suspension if difficult otherwise).
h) Centrifuge 10 min @ 15,000 x g.
4) Initial purification.
a) Resuspend pellet in 40 ml SB (use the tissue grinder to make even suspension if difficult otherwise; SB = 8M urea, 50 mM NaH2PO4, 0.3 M NaCl, pH 8.0). Stir for 1 hour.
b) Centrifuge 10 min @ 15,000 x g.
c) Apply supernatant to a NiNTA column equilibrated with 200 mL SB (use 5.0 mL resin per liter of cells).
d) Wash the column with 150 mL SB.
e) Elute the protein using a 600 mL (total volume) 0 to 0.3 M imadazole gradient in SB. Monitor the UV absorbance of the eluate using the ISCO flow cell (set to 2.0 AUFS).
f) Fractions corresponding to His6-TGFb3 are pooled. Solid DTT is added to give a final concentration of 0.25 M. Stir 30 min in a parafilm covered flask.
g) Dialyze 5 x 4 hr (4 C) against 4.0 L of 0.1 M HOAc. Typically observe little or no precipitate.
h) Concentrate to a volume of about 20 mL using an Amicon stirred cell.
5) Refolding
a) Estimate the amount of protein present by recording the UV absorbance (use 0.1 M HOAc as a blank). Assume 1 A280 is equivalent to 0.61 mg/mL.
b) Add concentrated fractions dropwise to enough 100mM Tris, 30mM CHAPS, 1M NaCl, 5mM reduced glutathione and 20% (v/v)
DMSO at 13 degrees and pH 9.5 while stirring gently to have a final TGF-b3 concentration of 0.2 mg/mL.
b) Continue stirring for 48 hours uncovered at 13C.
c) Dilute the solution approximately two-fold using MQ water.
e) Concentrate to ~ 100 mL using either an Amicon stirred cell or the Vivascience tangential flow concentrator.
f) Dialyze 3X versus 4L 0.1 M HOAc @4C.
g) Centrifuge 10 min. at 15,000 x g.
h) Filter supernatant through a 0.45 uM filter.
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) Elute with a linear gradient over 10 column volumes from 0% to 90% BufferB (Buffer A + 1M NaCl).
d) Typically see two peaks; the larger comes at about 41 % B and is the monomer; the smaller comes at about 52% B and is the dimer.
e) Fractions corresponding to the dimer are pooled and then dialyzed against 0.1 M HOAc.
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