| Geng S, Chang H, Qin W, Li Y, Feng J, Shen B.
(2008)
Preparative biochemistry and biotechnology,
38,
74-86 |
| Over expression & Renaturation |
| C-terminal hexahistidine tag |
| Protein recombinantly expressed as and refolded from inclusion bodies. |
| Escherichia coli |
| BL21(DE3) |
| 37.0 |
| 12 h |
| pET22b |
| Expression and Purification of the Inclusion Body
The recombinant expression plasmids were transformed
into BL21 (DE3) competent cells. The transformant
cells were grown at 37°C in 2×YT medium with 100 µg/mL
ampicillin. When OD600 reached 0.8, IPTG was added to
the final concentration of 1 mM to induce the
expression of the recombinant proteins. 12 h later,
the cells were harvested by centrifugation and
suspended in 10% PBS. The cells were sonicated at an
intensity of eight 50-s bursts on ice with a 50-s
cooling period between each burst. The particulates
were collected by centrifugation for 15 min at
10,000 ×g to remove soluble proteins. Then, a series
of washing buffers containing different concentrations
of urea (1 M, 2 M, 3 M, or 4 M urea, 5 mM EDTA, buffer
A, pH 7.4) was used to wash the inclusion bodies. |
| IPTG |
| OD 0.8 =
600 |
| Sonication |
| None |
| None |
| insoluble |
| Dilution |
| 1 M, 2 M, 3 M, or 4 M urea, 5 mM EDTA, buffer A, pH 7.4, EDTA, buffer A |
| 8 M urea, 10 mM -mercaptoethanol, 5 mM |
| 0.2% Tween 80, 5 mM EDTA, 0.8 mM, GSH/0.2 mM GSSG, buffer A |
| None |
| no |
| 0.0 |
| 4.0 |
| n/a |
| 48 h |
| GSH/GSSG |
| 0.8 mM- 0.2 mM |
| Refolding of the Inclusion Bodies
Renaturation is accomplished by the removal of excess
denaturants by dilution. The pH for each of the three
scFvs was unique and invariable in the process of the
denaturing, refolding, and dialyzing. According to the
pI of each scFv, the pH was decided, respectively.
The inclusion bodies were solubilized in denaturing
buffer (8 M urea, 10 mM -mercaptoethanol, 5 mM EDTA,
buffer A). The initial concentration of the inclusion
bodies was adjusted to 5 mg/mL. Then, the protein was
slowly diluted 25-fold into ice-cold refolding buffer
(0.2% Tween 80, 5 mM EDTA, 0.8 mM GSH/0.2 mM GSSG,
buffer A). The refolding mixture was stirred in the
process of diluting. After 48 h at 4°C, the samples
were dialyzed against dialyzing buffer (0.2% Tween 80,
1 mM EDTA, buffer A) twice for 12 h to remove the urea
and glutathione. |
| Enzyme activity |
| None |
| Tween 80 |
| 0.2% |
| 50% |
| n/a |
| Refolding notes: in this study they used optimized
refolding condition with different pH |