| Leong SS, Middelberg AP
(2007)
Biotechnology and Bioengineering,
97,
99-117 |
| Recombinant Protein Expression |
| None |
| Protein recombinantly expressed as and refolded from inclusion bodies. |
| Escherichia coli |
| BL21(DE3)RIL |
| 37.0 |
| 1 h |
| EP334-001 pET24D |
| rhAFP Expression
Recombinant E. coli strain BL21(DE3)RIL containing
plasmid EP334-001 pET24D 3.1A expressing rhAFP was
kindly provided by Merrimack Pharmaceuticals. It was
cultivated in 500 mL of 2YT media containing 50 mg/mL Kanamycin (GIBCO, Melbourne, Australia, 11815-032) in a 2 L baffled flask at 378C and 180 rpm using a shaking
incubator. Induction of rhAFP expression was done by
adding IPTG (Progen Biosciences, Brisbane, Australia, 200–
0121) to a final concentration of 0.4 mM when OD600
reached 1. Expression was terminated after 1 h of induction
at a final OD600 of 1.8 to 2. The final cell suspension was
divided into 250 mL aliquots and each aliquot centrifuged
(4,000g, 48C, 20 min). Cell pellets were washed in
phosphate-buffered saline (PBS: pH 7.2) and centrifuged
as above. Washed cell pellets were stored at
208C (for a maximum period of 2 weeks), and thawed prior to protein
extraction or mechanical disruption.
|
| IPTG |
| OD 1.8-2.00 =
600 |
| French Press |
| None |
| Affinity chromotography |
| insoluble |
| Dilution |
| 4 mM urea, 0.5% Triton X-100, 20 mM Tris, 3 mM EDTA, 10 mM DTT, pH 7.5 |
| 8 mM urea, 20 mM Tris, 3 mM EDTA, 20 mM DTT, pH 9 |
| 8 M urea, 20 mM Tris, 3 mM EDTA, 0.5 Arginine, 1,33 GSH, 1.33 GSSG, pH 8.5 |
| Affinity chromotography |
| no tag |
| 8.5 |
| 4.0 |
| n/a |
| 24 |
| GSH/GSSG |
| 1.33 mM,1.33 mM |
| Dilution Refolding
rhAFP fractions from the 100% Buffer D step elution of the
Phenyl Sepharose purification step were pooled for
refolding. DTT was first removed from the protein mixture
using a PD-10 desalting column containing Sephadex G-25
medium (GE Healthcare), equilibrated in Denaturing Buffer
(8 M urea, 20 mM Tris, 3 mM EDTA, pH 8.5). Although not
essential, this step ensures precise control of the redox
environment during refolding. In process implementation,
DTT could be easily omitted from Buffer D, but here it
was included to allow intermediate protein storage. PD-10
exchange step caused an approximate 1.4-fold dilution of
the protein mixture; actual protein concentration following
PD-10 exchange was determined by RP-HPLC (see
‘‘Analytical Methods’’). The buffer-exchanged denatured
protein mixture was rapidly diluted 10-fold into Refolding
Buffer (20 mM Tris, 3 mM EDTA, 0.5 M L-Arginine,1.33 mM GSH, and 1.33 mM GSSG, pH 8.5) in a single step.
The final Refolding Buffer composition was 20 mM Tris,
3 mM EDTA, 0.5 M L-Arginine, 0.8 M urea, 1.33 mM GSH,
and 1.33 mM GSSG, pH 8.5. The protein was incubated in
Refolding Buffer at 48C for 24 h. Aliquots (100 mL) of
samples were subsequently RP-HPLC analyzed to determine
the yield of correctly folded protein.
|
| HPLC |
| None |
| L-Arginine |
| 0.5 mM |
| n/a |
| n/a |
|