| Tanaka T, Kimura H, Hayashi M, Fujiyoshi Y, Fukuhara K, Nakamura H
(1994)
Protein Science,
3,
419-427 |
| Folding,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 |
| pGH-TIM |
| 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 10microg/ml final concentration and incubated a further 20h. |
| 3-beta-indoleacrylic acid |
| OD n/a =
n/a |
| Sonication |
| None |
| HPLC |
| insoluble |
| Dialysis |
| n/a |
| 20mM TrisHCl pH 8, 6M GdnHCl, 1% 2-mercaptoethanol |
| 50mM TrisHCl pH 7 |
| HPLC |
| yes |
| 7.0 |
| 4.0 |
| n/a |
| n/a |
| Beta-mercaptoethanol |
| 1% |
| Cells were harvested and disrupted by sonication. The insoluble fraction was collected by centrifugation and dissolved in solubilization buffer. After the insoluble materials were removed by centrifugation, the protein was precipiated by dialysis against 50mM TriHCl at pH7. The precipitate was dissolved in 70% formic acid containing 0.3M BrCN and treated for 2h to remove the fusion partner. The mixture was then subjected to revers HPLC. The purified protein was then dissolved in 50mM TrisHCl pH 7, 6M GdnHCl. The protein was then refolded by stepwise dialysis against refolding buffer containing 4, 3, 2, 1, and 0M GdnHCl. |
| Far-UV Circular Dichroism,Fluorescence,1H chemical shift (ppm),Gel filtration chromatography,Amino acid sequencing |
| None |
| None |
| n/a |
| n/a |
| n/a |
| n/a |