| Jin T, Guan YX, Yao SJ, Lin DQ, Cho MG.
(2006)
Biotechnol Bioeng,
93,
755-60 |
| Protein refolding |
| None |
| Protein recombinantly expressed as and refolded from inclusion bodies. |
| Escherichia coli |
| DH5a |
| 30.0 |
| 6 h |
| pBV220 |
| Several colonies of recombinant genetically engineered (pBV220/IFN-gDH5a) E. coli were obtained after growth on LB agar plates. A single colony was inoculated into 20 mL LB medium containing 100 mg/mL ampicillin in 250 mL shaking flask and cultivated at 308C and 200 rotation/min. After incubation overnight, the culture was transferred into a 3.7 L bioreactor (KLF2000, BioEngineering, Wald, Switzerland) with 2 L of 5􏰀 M9 þ LB sterile medium (30 g tryptone, 15 g yeast extract, 25.2 g Na2HPO4􏰁 12H2O, 6 g KH2PO4, 10 g NaCl, 2 g NH4Cl, adding fresh 2 mL of 1 mol/L MgSO4, and 30 mL of 20% glucose before use)
containing ampicillin 100 mg/mL. The cell cultivation was conducted at 308C, pH 6.8, and 400 rotation/min of agitating speed. When OD600 reached to about 0.8, the temperature was increased to 428C for inducing rhFN gamma expression. The cultivation was continued for 6 h. The cells were then harvested by centrifugation at 3.300 and 4c for 10 min. The collected cells were suspended in 200 mL lysis buffer and centrifuged as before. the washed cells were stored at -20. |
| Temperature Shift |
| OD 0.8 =
600 |
| ultrasonic homogenization |
| Detergents |
| None |
| insoluble |
| Column refolding-Expanded bed adsorption chromatography (EBA) |
| Lysis buffer (1 mmol/L EDTA, 50 mmol/L PBS, pH 7.0 ) |
| 8 mol/L urea, 10 mmol/L DTT, 1 mmol/L EDTA, 50 mmol/L PBS, pH 7.0 |
| A (8 mol/L urea, 50 mmol/L PBS, pH 7.0) B1 (50 mmol/L PBS, pH 7.0) B2 (1 mol/L NaCl, 50 mmol/L PBS, pH 7.5) |
| None |
| no tag |
| 7.5 |
| 0.0 |
| n/a |
| n/a |
| None |
| n/a |
| Expanded bed adsorption was conducted using a home-made column of 2.0 cm diameter. A small amount of glass beads (0.3 mm diameter, <5% total sedimented bed height) were added to improve flow distribution at the column inlet. The column was packed with 50 mL STREAMLINE SP for refolding experiments. The feedstock was conveyed by peristaltic pump (B. Brown, Germany). Three kinds of refolding buffers (see Table I) were used to perform the rhIFN-g refolding with a linear gradient of urea concentration. Prior to sample application, the EBA column was equilibrated with buffer A and the bed expansion was kept at the range of 2 – 3. UV Detector (K-2600, Knauer, Germany) was used to detect protein real-time. After the sample loading, the column was rinsed with buffer A until UV baseline was reached. The refolding of bound protein was performed in expanded bed mode by gradually increasing the ratio of buffer B1/ buffer A to form a linear gradient of urea concentration from 8 mol/L to a defined low concentration. After the refolding process was completed, the refolded protein was eluted with buffer B2 on packed bed mode. Finally, STREAMLINE SP was cleaned-in-place (CIP) and regenerated by 0.5 mol/L NaOH and 1 mol/L NaCl for recycle use. |
| Antibacterial activity assay |
| None |
| None |
| n/a |
| n/a |
| n/a |
| refolding temperature is not stated |