| Han YG, Liu HL, Zheng HJ, Li SG, Bi RC.
(2004)
Protein Expression and Purification,
35,
360-365 |
| Protein refolding |
| N-terminal hexahistidine tag |
| Protein recombinantly expressed as and refolded from inclusion bodies. |
| Escherichia coli |
| BL21(DE3) |
| 37.0 |
| 3 h |
| pET21b |
| The plasmid was provided by Shanghai Institute of Hematology, Rui jin Hospital, Shanghai Second Medical University. As shown in Fig. 1, the gene encoding the human PSMA5 was subcloned into the vector pET-22b (+) at the sites cut by NdeI and XhoI and sequenced. The sequence encoding 6× histidine is downstream to the sequence encoding the human PSMA5, which was expressed as a 6× His-tagged fusion protein. The induction procedure proved critical to achieve the highest recombinant quantity or activity yields. Isolated colonies of transfected E. coli BL21 (DE3) were inoculated into LB medium (2.5 mL, 100 μg/mL ampicillin) and grown at 37 °C. When the OD600 reached 0.6, the cell suspension was transferred to 15 mL LB medium and grown at the same conditions. Finally, cells were transferred to 500 mL LB medium and grown until the OD600 reached 0.6. 1 mM IPTG (isopropyl-β-thiogalactopyranoside) was subsequently added and the suspension was incubated at 37 °C for 3 h. |
| IPTG |
| OD 0.6 =
600 |
| Sonication |
| Detergents |
| Affinity chromotography |
| insoluble |
| Column refolding: Size-exclusion chromatography |
| 8% sucrose, 10 mM Tris–HCl, and 2% Triton X-100, pH 8.0 |
| 50 mM Tris–HCl, 300 mM NaCl, 5 mM imidazole, and 8 M urea, pH 8.0 |
| 5 mM EDTA, 2 mM DTT, 1 mM oxidized glutathione, 5 mM reduced glutathione, 0.1 mM PMSF, and 50 mM Tris–HCl, pH 7.3 |
| Affinity chromotography |
| no |
| 7.3 |
| 25.0 |
| n/a |
| n/a |
| GSH/GSSG/DTT |
| 5/1/2 mM |
| Size-exclusion chromatography refolding
The experimental procedures for chromatography refolding have special virtues [13]. Denatured recombinant protein was refolded using Sephadex 75 HR 10/30 column (Pharmacia). The human PSMA5 was first filtered through a 0.45 μm Millipack cellulose acetate filter (Millipore) to remove particulates. Four milliliters of denatured protein was injected onto the column, which was previously equilibrated with 3–5 column volumes refolding buffer. The human PSMA5 was eluted at the rate of 1 mL/min using refolding buffer as the mobile phase. Absorbance at 280 nm was monitored with a UV detector connected to a data acquisition package. Experiments were carried out at room temperature and in duplicate. |
| Gel filtration chromatography |
| None |
| None |
| n/a |
| 20% |
| 86% |
| n/a |