| Khan RH, Rao KB, Eshwari AN, Totey SM, Panda AK.
(1998)
Biotechnol Prog,
1998,
722-728 |
| Over expression & Renaturation |
| N-terminal hexahistidine tag |
| Protein recombinantly expressed as and refolded from inclusion bodies. |
| Escherichia coli |
| m15 |
| 37.0 |
| 5-6 h |
| pQE-30 |
| The fermentation was carried out at 37 °C with vigorous aeration and agitation and at OD600 ) 10 were induced with 1 mM IPTG and cultivated for another 5-6 h. The harvested cells were checked for expression and processed for the purification of inclusion bodies. Samples were collected at an interval of 30 min during the fermentation to check cell growth, glucose consumption, and protein expression. For the analysis of protein concentration during different stages of growth, inclusion bodies were separated from the cell and purified and then dissolved in 1% SDS solution in 50 mM Tris-HCl buffer at pH 8. Protein content was determined by BCA assay and growth hormone levels by RIA. |
| IPTG |
| OD 10 =
600 |
| French Press |
| None |
| not specified |
| insoluble |
| Column refolding: Size-exclusion chromatography |
| 50 mM Tris-HCl buffer (pH 8.0) containing 5 mM EDTA and 2% deoxycholate solution |
| 2 M Tris buffer pH 12 and 2 M urea |
| 100 mM Tris-HCl (pH 8) |
| not specified |
| no |
| 8.0 |
| 25.0 |
| n/a |
| n/a |
| None |
| n/a |
| In the second method, refolding of the recombinant oGH was carried out in gel filtration chromatography using the recently reported method for refolding of denatured lysozyme and carbonic anhydrase (25). The main pur-
pose was to allow the solubilized protein to enter the gel filtration matrix and then carry out the buffer exchange, so that the intermolecular aggregation could be avoided. A Sephacryl S-100 column (16 × 800 mm) was equilibrated with 100 mM Tris-HCl (pH 8) with a flow rate of 30 mL/h. Solubilized proteins were loaded into the column, and the eluted fractions were monitored with an UV detector and further analyzed by SDS-PAGE and
Western blotting. Refolding experiments were carried out within 2 h of the solubilization.
|
| Western Blot,SDS-PAGE,Receptor binding,Circular Dichroism (uv-CD) |
| None |
| None |
| n/a |
| 60-70% |
| n/a |
| n/a |