| Pelleted insoluble material was resuspended in 35 mLof 100 mM Tris-HCl, pH 7.0, 5 mM EDTA, 10 mM DTT, 2 M urea, 2% v/v Triton-X 100 per liter of starting fermentation product. The suspension was placed on ice and subjected to sonication high power with 2 min intervals, followed by centrifugation ~13,500 rpm, 4 8C! for 30 min. This wash method was repeated two
additional times. After the third wash, the pelleted material was resuspended in 25 mL of 100 mM Tris-HCl, pH 7.0, 5 mM EDTA, 10 mM DTT per liter of starting fermentation product, sonicated
for 1 min at 4 8C and centrifuged ~13,500 rpm, for 30 min. This second wash step was also repeated twice. The washed inclusion
bodies were pelleted as above.
The washed inclusion bodies were solubilized by the addition of 10 mL 6 M guanidinium chloride, 0.1 M NaH2PO4, 10 mM Tris-HCl, pH 8.5. To completely solubilize inclusion bodies, the
suspension was placed on ice and subjected to sonication for 1 min at high power. The solution was centrifuged at 18,000 rpm for 15 min to separate insoluble material. The solution was reduced by the addition of 20 mM DTT and allowed to stand at 37C for 30 min.
A 50 mL metal chelating column was prepared using Chelating Sepharose Fast Flow resin. The column was washed with three column volumes H2O, followed by five column volumes
of 0.1 M NiCl2. A further three column volumes of H2O followed by three column volumes of 6 M GdCl, 0.1 M NaH2PO4, 10 mM Tris-HCl, pH 8.5 were used to equilibrate the column. The
reduced protein solution was loaded onto the column at 10 mL0 min. The column was then washed with five column volumes of 8 M urea, 0.1 M NaH2PO4, 10 mM Tris-HCl, pH 8.5, followed by five column volumes of 8 M urea, 0.1 M NaH2PO4, 10 mM Tris-HCl, pH 6.3. The bound fraction was eluted with 6 to 10 3 5 mL volumes of 8 M urea, 0.1 M NaH2PO4, 10 mM Tris-HCl,
0.5 M imidazole, pH 5.9. Pooled fractions from Ni21 purification were reduced with 40 mM
DTT for 1 to 2 h. A 50 mL SP-Sepharose column was prepared by equilibrating with five column volumes of 6 M urea, 10 mM NaH2PO4, 10 mM Tris-HCl, pH 5.8 ~Buffer A!. The protein solution
was diluted threefold with Buffer A and loaded onto the column at 10 mL0min. Full-length monomeric VEGF-B167 was separated from the truncated form using a linear gradient formed
between Buffer A and 6 M urea, 10 mM NaH2PO4, 10 mM Tris-HCl, 1 M NaCl, pH 5.8 (Buffer B). Fractions containing full-length VEGF-B167 were pooled and diluted
to 0.1 mg/mL with 6 M urea, 0.1 M NaH2PO4, 10 mM Tris-HCl, 1 mMEDTA, 20 mM DTT, pH 8.5, then dialyzed against 100 mM Tris-HCl, 5 mM cysteine, 1 mM cystine, 0.5 M GdCl,
2 mM EDTA, pH 8.5, overnight at room temperature. The solution was then dialyzed against 0.1 M acetic acid for approximately 24 h
and filtered through a 0.22 mM cellulose acetate filter, to remove particulate matter.
To separate dimeric VEGF-B167 from mono- and multimeric species the acidified protein solution was diluted fivefold with 80% v/v n-propanol, 10 mM NaCl, pH 2.0 (Buffer C), and loaded
onto a Polyhydroxymethyl A hydrophilic column (PolyLC). A linear gradient was formed between Buffer C and 10 mM NaCl, pH 2.0 (Buffer D). Fractions containing dimeric VEGF-B167 were
combined, diluted fourfold with water, and loaded onto a C8 reversed-phase column pre-equilibrated at 45C with 0.15% TFA ~Buffer E! using a GOLD Beckmann system. A linear gradient was formed between Buffer E and 0.13% v/v TFA, 60%
v/v acetonitrile ~Buffer F!. Fractions containing dimeric VEGFB167 were pooled, diluted, and reapplied to the C8 column. The protein was eluted with Buffer F to minimize sample dilution. |