| 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 BodiesIn 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 |
| Dilution |
| 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 |
| 0.2 M l-arginine, 1.0 mM EDTA, and 20 mM PB, pH 7.4 |
| None |
| no tag |
| 7.4 |
| 15.0 |
| 10.0 mg/ml |
| 1 h |
| None |
| n/a |
| Solubilization (Denaturation) of Inclusion Bodies, Ten grams of IBs were dissolved in 200 ml of dissolved buffer containing 6 M guanidine–HCl (Gu-HCl), 50 mM phosphate buffer (PB), 100 mM NaCl, and 5 mM EDTA, pH 7.4, at room temperature for 60 min with gentle stirring. Insoluble material was removed by centrifugation at 10,000 rpm and 4 °C for 30 min. The supernatant containing 10.0 mg/ml protein was immediately subjected to dilution or size-exclusion chromatography.Protein Refolding by Dilution, Process optimization on the renaturation of Y1-Sak was carried out using the dilution method. The effects of various factors, including pH, temperature, initial concentration of Y1-Sak, and small molecular additives, on the refolding efficiency of Y1-Sak were studied in detail. In brief, a 0.1-ml sample of isolated and dissolved IBs (10 mg/ml in 6 M Gu-HCl) was diluted 100-fold in the PB and kept at 15 °C without stirring for 1 h. |
| Mass spectrometry,Circular Dichroism (uv-CD) |
| None |
| L-Arginine |
| 0.2 M |
| 40%-50% |
| 85% |
| n/a |