| Li M, Bullock CM, Knauer DJ, Ehlert FJ, Zhou QY.
(2000)
Mol Pharmacol.,
59,
692-8 |
| Identification and Characterization |
| C-terminal hexahistidine tag+GST tag |
| Protein recombinantly expressed as and refolded from inclusion bodies. |
| Escherichia coli |
| BL21 |
| 37.0 |
| 2 h |
| pGEX-3X |
| The coding sequences for mature prokineticins were cloned into the prokaryotic expression vector pGEX-3X (Amersham Pharmacia Biotech, Piscataway, NJ). The extra nucleotides between the factor Xa protease digestion site of the glutathione-S-transferase (GST) tag and mature prokineticins were removed by site-directed mutagenesis and confirmed by sequencing. To facilitate protein purification, a 6xHis-tag was added to the C terminus so that the fusion proteins could be purified with Ni-NTA affinity chromatography (Qiagen, Valencia, CA). The detailed protocols for production of fusion proteins are as follows. Escherichia coli cells (BL21) were grown to absorbance 0.8 and induced with 600 µM isopropyl -D-thiogalactoside for 2 h at 37°C. The cells were then pelleted, washed, and lysed with buffer A (6 M guanidine hydrochloride, 100 mM NaH2PO4, and 10 mM Tris, pH 8.0). Fusion proteins were allowed to bind to Ni-NTA beads and then washed extensively with buffer C (8 M urea, 100 mM NaH2PO4, and 10 mM Tris, pH 6.3) and buffer D (8 M urea, 100 mM NaH2PO4, and 10 mM Tris, pH 5.9). |
| IPTG |
| OD n/a =
n/a |
| Bead Beater |
| None |
| Ni-NTA agrose chromatography |
| insoluble |
| Dilution/Dialysis combination |
| 8 M urea, 100 mM NaH2PO4, and 10 mM Tris, pH 5.9 |
| 50 mM Tris, 150 mM NaCl, and 1 mM CaCl2, pH 7.5 |
| 4 M urea, 5 mM cysteine, 0.02% Tween-20, 10% glycerol, 10 mM Tris, 150 mM NaCl, 100 mM NaH2PO4, pH 8.3 |
| Ni-NTA agrose chromatography |
| yes |
| 4.5 |
| 25.0 |
| n/a |
| n/a |
| Cysteine |
| 5 mM,5 mM |
| The cells were then pelleted, washed, and lysed with buffer A (6 M guanidine hydrochloride, 100 mM NaH2PO4, and 10 mM Tris, pH 8.0). Fusion proteins were allowed to bind to Ni-NTA beads and then washed extensively with buffer C (8 M urea, 100 mM NaH2PO4, and 10 mM Tris, pH 6.3) and buffer D (8 M urea, 100 mM NaH2PO4, and 10 mM Tris, pH 5.9). Fusion protein-bound beads were equilibrated with digestion buffer (50 mM Tris, 150 mM NaCl, and 1 mM CaCl2, pH 7.5). Digestion was performed overnight at room temperature with 10 ng of protease factor Xa per microgram of fusion protein. The cleaved GST tag was then washed away with buffer D. Mature prokineticins were eluted with buffer E (8 M urea, 100 mM NaH2PO4, and 10 mM Tris, pH 4.5), and fractions were analyzed by SDS-PAGE. The pooled recombinant prokineticins were then refolded as follows. Proteins were diluted to 100 µg/ml with buffer E and dialyzed against renaturing buffer (4 M urea, 5 mM cysteine, 0.02% Tween-20, 10% glycerol, 10 mM Tris, 150 mM NaCl, 100 mM NaH2PO4, pH 8.3). New renaturing buffer (same as above except with 2 M urea) was then added, and dialysis was continued for 4 more days with at least one more change of renaturing buffer. The refolded protein was then desalted with a spin column (Qiagen) and analyzed by receptor binding or bioassay. The final purification was performed with reverse phase-HPLC (Amersham Pharmacia Biotech). Functional proteins were eluted with 0.08% trifluoroacetic acid and a 10 to 50% acetonitrile gradient. The elution of protein was monitored at 206 nm. Trifluoroacetic acid and acetonitrile were then evaporated by lyophilization. |
| Receptor binding,Mass spectrometry |
| None |
| Glycerol,(NH4)2SO4,tween 20 |
| 100mM/10%/0.02% |
| n/a |
| n/a |
| n/a |