| Kim KT, Mok MT, Edwards MR.
(2005)
Biochemical and Biophysical Research Com,
334,
333-341 |
| Identification and Characterization |
| None |
| Protein recombinantly expressed as and refolded from inclusion bodies. |
| Escherichia coli |
| None |
| 0.0 |
| 00 |
| pQE-30 |
| Expression of the rGiPKB. A 1.8 kb KpnI/BamHI fragment containing the GiPKB ORF was amplified from genomic DNA by Pfu DNA polymerase (Promega) and primers GiPKBFor and GiPKBRev. This fragment was subsequently cloned into a pQE-30 expression vector (Qiagen) followed by transformation into Escherichia coli cells. Cells were grown according to the manufacturer’s instructions and harvested by centrifugation. The cell pellet was washed once in PBS and resuspended in Tris buffer (10 mM Tris buffer, pH 7.4, 1 mM E-64), followed by sonication using a Branson Sonifier 250. |
| Not Stated |
| OD n/a =
n/a |
| Sonication |
| None |
| Metal affinity chromatography |
| insoluble |
| Dilution |
| PBS |
| Tris buffer containing 6 M urea |
| Tris buffer (10 mM Tris, pH 7.4, 0.2 mM EDTA, 1% Triton X-100, and 10% glycerol) |
| Metal affinity chromatography |
| no |
| 7.4 |
| 0.0 |
| n/a |
| n/a |
| None |
| n/a |
| Purification and renaturation of the rGiPKB. Purification of the rGiPKB was performed using a His-trap chelating column (Pharmacia) according to the manufacturer’s instructions unless otherwise specified. Unbound proteins were removed by washing with 100 mM imidazole buffer, prior to elution of rGiPKB using 500 mM imidazole buffer. The denatured protein was renatured by a serial dilution with Tris buffer (10 mM Tris, pH 7.4, 0.2 mM EDTA, 1% Triton X-100, and 10% glycerol). Purified recombinant and total cellular proteins were quantified using the Bio-Rad protein assay. |
| Western Blot,SDS-PAGE,Protein kinase assay |
| None |
| Glycerol,Triton X-100 |
| 10%/1% |
| n/a |
| n/a |
| n/a |