| Birch GM, Black T, Malcolm SK, Lai MT, Zimmerman RE, Jaskunas SR
(1995)
Protein Expression and Purification,
6,
609-618 |
| Functional Studies |
| None |
| Protein recombinantly expressed as and refolded from inclusion bodies. |
| Escherichia coli |
| RV308 |
| 42.0 |
| 3h |
| p3ABC |
| For large-scale expression, 100L of cells were grown in LB media with tetracycline at 30degC in a fermentor with pH controlled at 7.0 with dissolved oxygen level of 50% or greater. For induction, cells were increased to 42degC and the cells were incubated for 3h. Cells were harvested by centrifugation and stored at -70degC. |
| Temperature Shift |
| OD =
|
| High pressure homogenization |
| Lysozyme |
| Ion-exchange chromatography |
| soluble |
| Dialysis |
| 1.0M NaCl, 1.0M deionized urea, |
| 7M urea, 50mM Tris-acetate, 1mM EDTA, 20mM cysteine, pH 7.5 |
| 20mM MES, 1mM EDTA, 1mM DTT, pH 6.5 |
| Ion-exchange chromatography |
| no tag |
| 6.5 |
| 4.0 |
|
|
| DTT |
| 1mM |
| The cell paste (~200g) was resuspended at room temperature with 1800ml of 50ml TrisHCl pH 8.0. Cells were lysed by the addition of lysozyme (200ml of 4mg/ml in 50mM TrisHCl, 50mM EDTA pH 8.0) and homogenized. The cells were centrifuged (10000g x 45min), and the pellet was washed several times with at 4degC wash buffer, then stored at -20degC. The inclusion bodies were thawed and resuspended in solubilization buffer, then centrifuged (30min x 30000g). The supernatant was then loaded onto a Q-sepharose Fast Flow column and eluted in solubilization buffer at 6.0ml/min. Selected fractions were collected and loaded onto a SP sepharose HP cation-exchange column equilibrated with 7M urea, 30mM TrisHCl, 1mM EDTA, 20mM cysteine pH 8.0 (Buffer B). The protein was eluted with a linear gradient over 10 column volumes of buffer B + 300mM KCl. Selected fractions were pooled, DTT was added to a 100x (mol/mol) ratio to protein, then the protein was dialyzed against refolding buffer. The refolding mixture was centrifuged (30000g x 20min) and the supernatant (~2.5mg/ml) was concentrated to ~7.5mg/ml. ~75mg of protein was then loaded onto a Superdex75 size exclusion column and eluted with refolding buffer. Selected fractions were pooled and glycerol added to a final concentration of 10%. The protein was stored at -70degC.
|
| HPLC |
| None |
| None |
|
| 60mg/mL cell culture |
|
|