| Tomasselli AG, Paddock DJ, Emmons TL, Mildner AM, Leone JW, Lull JM, Cialdella JI, Prince DB, Fischer HD, Heinrikson RL, Benson TE.
(2008)
Protein Pept Lett.,
15,
131-143 |
| Protein refolding |
| None |
| Protein recombinantly expressed as and refolded from inclusion bodies. |
| Escherichia coli |
| BL21 |
| 37.0 |
| 3 h |
| pET11a, pQE80L, pQE70 |
| Protein Expression
Protein expression was essentially the same for the three
constructs. Cells were grown in Luria Broth (LB), pH 7.5,
with 100 μg/ml ampicillin and 34 μg/ml chloramphenicol, at
37°C and 200 rpm (2.5 inch throw). A loop full from a glyc-
erol stock of the construct was inoculated into the media and
was incubated until the A550 = 0.5 –0.6. Cells were collected
by centrifugation, resuspended in fresh media, and used as
inoculum for a secondary culture at a 1:100 dilution. When
cell density reached A550 = 0.5 - 0.6, cells were harvested by
centrifugation at room temperature and then resuspended at
the same concentration in fresh LB, again containing am-
picillin and chloramphenicol. BACE expression was induced
by the addition of IPTG to a final concentration of 1mM.
Expression of the recombinant protein was continued for 3
hours after induction (A550 = 1.8 - 2.0). Cells were collected
by centrifugation and stored at –80o C. Initially cultures had
been prepared in 100 ml of media contained in 500 ml flasks.
This process was easily adaptable to a larger flask size and
was scaled-up as follows. To shorten the overall length of the
process, the inoculation rate for the secondary culture was
increased 4-fold to a 1:25 dilution of the primary seed cul-
ture. Cultures were grown in 1.25 L of media contained in
2.8 L Fernbach flasks and the shaking rate was increased to
225 rpm. To further facilitate production of BACE, the trans-
fer of the culture to fresh media before induction was elimi-
nated. Other conditions remained identical to the 1.25 L
process, except the culture was grown to an A550 = 0.75 -
0.85 before induction with IPTG and expression was contin-
ued for only 2.5 hours.
Protein Localization
Cell paste was resuspended in TE buffer (10mM Tris HCl,
pH 8.0, 1mM EDTA) at 1/10 of the original culture
volume and sonicated. The soluble protein fraction was sepa-
rated from cell debris and insoluble proteins by centrifuga-
tion at 10,000 x g for 15 minutes. Fractions were analyzed by
SDS-PAGE.
|
| IPTG |
| OD 1.8-2.00 =
550 |
| French Press |
| None |
| None |
| insoluble |
| Dilution |
| 10mM Tris HCl, HCl, pH 8.0, 1 mM EDTA |
| 15-20 ml 8 M urea, 100mM BME ( B-mercaptoethanol ) |
| 20-25 fold with water (4-25°C; pH 9.5 to 11.5) |
| None |
| no tag |
| 9.5 |
| 4.0 |
|
| overnight |
| None |
| n/a,100 mM,n/a,n/a,n/a |
| Refolding
The solution with an A280 reading of 0.4-0.7 was quickly diluted 20-25 fold with water (4-25°C; pH 9.5 to 11.5). The sample was then allowed to stand in the cold room for several days. Activity could be measured within 24 hours of the 20-25 fold dilution step, and activity assays were performed daily to monitor the refolding process.
|
| Unspecified,Protein activity assay |
| None |
| None |
| n/a |
| n/a |
| n/a |
|