| Chan YH, Cheng CHK and Chan KM
(2003)
comparative Biochemistry and Physiology,
135,
613-624 |
| Recombinant Protein Expression |
| N-terminal hexahistidine tag |
| Protein recombinantly expressed as and refolded from inclusion bodies. |
| Escherichia coli |
| C41 (DE3) |
| 37.0 |
| 5 h |
| pRSETA |
| The transformed cells were grown in LB medium (500 ml) containing 50 ug/ml of ampicillin to an OD600 of approximately 0.4; IPTG was then added to a final concentration of 1 mM to induce specific protein expression for 5 h. Cells were collected by centrifugation at 4000xg for 10 min.
Inclusion bodies of the insoluble recombinant hormones were collected as described ([Binder et al]). In brief, cells from 500 ml culture were collected, washed and suspended in 25 ml cell suspension buffer (20 mM TrisHCl, pH 7.5; 50 mM EDTA). Lyzoyme was added to the cell suspensions at a final concentration of 0.1 mg/ml, followed by incubated at 37 C for 1 h with gentle shaking. Triton X-100 was further added to a final concentration of 1%, and kept at room temperature for 30 min with gentle shaking. The cells were disrupted by sonication and the insoluble fractions were collected by centrifugation at 18 000xg for 10 min. |
| IPTG |
| OD 0.4 =
600 |
| Sonication |
| Lysozyme |
| Metal affinity chromatography |
| insoluble |
| Dilution/Dialysis combination |
| 20 mM TrisHCl, pH 7.5; 50 mM EDTA |
| 20 mM TrisHCl, pH 7.5; 6 M urea |
| 50 mM TrisHCl (pH 9.0), 2 M urea, 20 mM mercaptoethanol and 2 mM oxidized glutathione (GSSG) |
| Metal affinity chromatography |
| no |
| 9.0 |
| 4.0 |
| n/a |
| overnight |
| GSSG/beta mercaptoethanol |
| 2 mM/ 20 mM,2-20 mM,2 mM/ 20 mM,2 mM/ 20 mM,2 mM/ 20 mM |
| The inclusion bodies were dissolved in 5 ml of dissolving solution (20 mM TrisHCl, pH 7.5; 6 M urea). Purification of the recombinant gfGHs carrying the 6XHis tag were partially purified by metal chelating affinity chromatography using a Ni2+-charged HiTrap™ chelating column (1 ml, Pharmacia) on the FPLC system (Pharmacia). The dissolved protein (2 ml) was loaded onto the column, after washing with 5 ml washing solution (20 mM sodium phosphate, pH 6.5; 0.5 M NaCl; 6 M urea), the bound proteins were eluted with 5 ml elution solution (20 mM sodium phosphate, pH 6.5; 0.5 M NaCl; 6 M urea; 50 mM EDTA). The eluted protein solution was gradually diluted (1:5) with a buffer containing 50 mM Tris¡VCl (pH 9.0), 2 M urea, 20 mM £]-mercaptoethanol and 2 mM oxidized glutathione (GSSG). Urea was gradually removed by dialysis against 25 mM ammonium bicarbonate overnight at 4 C to facilitate refolding of the recombinant gfGHs. After being clarified by centrifugation at 18 000g for 10 min, the protein solutions were then stored at ?70 C in aliquots for different assays.
|
| SDS-PAGE |
| None |
| None |
| n/a |
| 50-70 % |
| n/a |
| n/a |