| He J, Wang G, Xu R, Feng J, Wang J, Su H, Song H.
(2008)
Appl Biochem Biotechnol.,
151,
29-41 |
| Protein refolding |
| None |
| Protein recombinantly expressed as and refolded from inclusion bodies. |
| E coli |
| JF1125 |
| 42.0 |
| 2.5 h |
| pLY-4 |
| Inoculum was prepared by streaking sterile Luria–Bertani agar plates containing 50 μg of ampicillin per milliliter with cells obtained from the glycerol stock culture. A single colony from the plates was used to inoculate 5 ml of the Luria–Bertani/ampicillin medium and grown for 8 h at 30 °C and 200 rpm in a gyratory shaker. Then, 0.5 ml of this culture was transferred aseptically to 500 ml of the Luria–Bertani/ampicillin medium and cultured for another 8 h under the same conditions. Subsequently, the cells were transferred into 25 l of M9 medium supplemented with a mixture of 2% (w/w) glucose and 2% (w/w) casamino acids in a 30-l fermentor [14]. This culture was grown at 30 °C until the culture turbidity (optical density at 600 nm) reached 3.5. Then, the culture temperature rose swiftly from 30 °C to 42 °C, and the cells were grown at 42 °C for 2.5 h before harvest. Cells harvested from a 30-l fermentor were then either frozen at −20 °C or processed immediately. Isolation and Purification of Inclusion Bodies In a typical preparation, 50 g cell pellet was resuspended in 1,000 ml disruption buffer (50 mM phosphate-buffered saline [PBS], 5 mM ethylenediamine tetraacetic acid [EDTA], pH 7.4) and homogenized with high-pressure homogenizers (JHG-Q54-P70; Shanghai Zhangyan Light Industry Equipments). A total of three to five passes were performed to obtain at least 95% cell disruption. The precipitates were collected by centrifugation at 10,000 × g and 4 °C for 30 min. SDS-PAGE analysis showed that Y1-Sak was mainly in the pellet (data not shown). Therefore, Y1-Sak was expressed mostly in the form of IBs.Fifteen grams of crude IBs were washed three times with 50 ml disruption buffer containing 0.5% Triton X-100 in using a homemade homogenizer. The purified IBs were stored at −20 °C. |
| Temperature Shift |
| OD n/a =
n/a |
| High pressure homogenization |
| None |
| None |
| partial |
| Column refolding: Size-exclusion chromatography |
| 50 mM phosphate-buffered saline [PBS], 5 mM ethylenediamine tetraacetic acid [EDTA], pH 7.4, 0.5% Triton X-100 |
| 6 M guanidine–HCl (Gu-HCl), 50 mM phosphate buffer (PB), 100 mM NaCl, and 5 mM EDTA, pH 7.4 |
| PB pH 7.4 containing 0.2 M Gu-HCl |
| None |
| no tag |
| 7.4 |
| 24.0 |
| n/a |
| n/a |
| None |
| n/a |
| Size-exclusion refolding was performed using a column (2.5 cm diameter × 100 cm long) packed with Sephacryl S-100 gel media. Prior to sample application, the chromatographic apparatus was cooled to the chosen temperature. The Sephacryl S-100 column was first loaded with 0.75 column volumes of pH 7.4 buffer containing 0.2 M Gu-HCl followed by a gradient of 0.25 column volumes from 0.2 to 6 M Gu-HCl. When the sample was applied, the top of the column was in 6 M Gu-HCl, whereas from 0.25 column volumes downward, the Gu-HCl concentration was 0.2 M. After application of the sample and an additional small volume of high Gu-HCl buffer, the Y1-Sak was eluted with PB pH 7.4 containing 0.2 M Gu-HCl. As the proteins in the sample are excluded from the beads, they will flow through the column at a higher running speed than Gu-HCl, thus passing gradually from the denaturing to refolding buffer. The speed of this transition is governed by the flow rate. Thus, the effect of the flow rate on recovery of active Y1-Sak was investigated in the range of 0.5 to 2.5 ml/min. In addition, the effect of sample volume on the recovery of active fragment was studied in the range of 5.0 to 30.0 ml. In control experiments, the column was operated without the Gu-HCl gradient, and all other parameters were kept constant (sample volume 15 ml [10.0 mg/ml in 6 M Gu-HCl] and flow rate 1.5 ml/min).
The refolded protein was dialyzed against 0.02 M PB (pH 7.4) after size-exclusion chromatography. Then, the refolded protein was further purified by a Q Sepharose FF column (2.5 cm diameter × 10 cm long) pre-equilibrated with 10 mM PB, pH 7.4. After loading, the column was prewashed with about 1 bed volume of buffer to elute a small amount of unbound impurities. Then Y1-Sak was eluted with a linear gradient of 0–0.3 M NaCl in 20 mM PB (pH 7.4). The elution flow rate was 40 cm/h. The elution peak was detected at 280 nm. The elution peak was present at about 0.08 M NaCl, and elution fractions (120 ml) were collected. |
| Mass spectrometry,Circular Dichroism (uv-CD) |
| None |
| None |
| n/a |
| n/a |
| n/a |
| n/a |