| Tanaka T, Hayashi M, Kimura H, Oobatake M, Nakamura H
(1994)
Biophysical Chemistry,
50,
47-61 |
| Structural Studies |
| N-terminal human growth hormone portion (aa.1-122) |
| Protein recombinantly expressed as and refolded from inclusion bodies. |
| Escherichia coli |
| HB101 |
| 37.0 |
| 20h |
| not stated |
| Cells were grown in M9 medium containing a 2-fold strentgh of the salts and 0.5% casamino acids. When the culture had attained a density between 120-200 Klett units, protein expression was induced by the addition of 3-beta-indoleacrylic acid to 20microg/ml final concentration and incubated a further 20h. |
| 3-beta-indoleacrylic acid |
| OD n/a =
n/a |
| Sonication |
| Lysozyme |
| HPLC |
| insoluble |
| Dialysis |
| n/a |
| 6M GdnHCl, 1% 2-mercaptoethanol |
| 10mM sodium acetate, pH 4 |
| HPLC |
| yes |
| 4.0 |
| 4.0 |
| n/a |
| n/a |
| Beta-mercaptoethanol |
| 1% |
| Cells were harvested and suspended in lysis buffer (50mM TrisHCl pH 7.5, 100mM NaCl, 1mM EDTA). After addition of lysozyme and standing at 0degC for 30min, the mixture was sonicated (4x20sec). The insoluble fraction was collected by centrifugation and dissolved in solubilization buffer. After the insoluble materials were removed by centrifugation (1h, 4degC, 5000g), the protein was precipiated by dialysis against H2O. The precipitate was collected and treated with brCN in 80% HCOOH for 3h to cleave the fusion partner. The mixture was then subjected to reverse HPLC. The purified protein was then dissolved in 10mM sodium acetate pH 4 with 8M GdnHCl. The protein was then refolded by stepwise dialysis against refolding buffer containing 6, 4, 2 and 0M GdnHCl. |
| Far-UV Circular Dichroism,Fluorescence,1H chemical shift (ppm),Gel filtration chromatography,Analytical centrifugation |
| None |
| None |
| n/a |
| n/a |
| n/a |
| n/a |