| Guo YW, Chang TY, Lin KT, Liu HW, Shih KC, Cheng SH.
(2001)
Protein Expression and Purification,
23,
483-490 |
| Cloning and expression |
| N-terminal hexahistidine tag |
| Protein recombinantly expressed as and refolded from inclusion bodies. |
| Escherichia coli |
| BL21(DE3) |
| 0.0 |
| 2 h |
| pET21a |
| The expression plasmid was constructed by ligating the coding region of the mucrosobin cDNA into a pET21a vector (Novagen Inc., WI) at the BamHI and EcoRI sites. Plasmid containing a 0.7-kb DNA insert, designated as pMucrosobin, was then introduced into Escherichia coli BL21(DE3) cells for protein expression.Expression and Purification of Recombinant Protein BL21(DE3) cells containing pMucrosobin were grown 32 to a late log phase (A600 =0.3–0.4) in Luria–Bertani broth and induced with 1 mM isopropyl-B-D-thiogalactoside (IPTG) for 2 h. The cells were pelleted and sonicated in an 8 M urea buffer (8 M urea, 0.1 M NaH2PO4, 10 mM Tris –HCl, pH 8.0). The resulting lysate containing the recombinant mucrosobin protein was incubated with Ni-NTA affinity resin at 4C for 2 h. The resin was then washed twice with the 8 M urea buffer. The recombinant protein was eluted from the resin with the same buffer containing 400 mM imidazole. The yield of the purified protein was 8 mg/liter of bacterialculture. |
| IPTG |
| OD 0.3-0.4 =
600 |
| Sonication |
| None |
| not specified |
| insoluble |
| Dilution |
| n/a |
| 8 M urea, 0.1 M NaH2PO4, 10 mM Tris –HCl, pH 8.0 |
| 50 mM Tris –HCl, pH 8.0/0.1 mM EDTA/0.75 M NaCl/0.01% Tween 20/20% glycerol/ 1 mM oxidized glutathione and 2 mM reduced glutathione/0.3 M urea (see notes) |
| not specified |
| no |
| 8.0 |
| 22.0 |
| n/a |
| 24 h each step |
| GSH/GSSG |
| 2/1 mM |
| Refolding and Characterization of Recombinant Protein Expressed in E. coli Purified mucrosobin protein was adjusted to a final concentration of 5 ug/ml protein. Diluted protein was refolded first in buffer I (50 mM Tris –HCl, pH 8.0/0.1 mM EDTA/0.75 M NaCl/0.01% Tween 20/20% glycerol/ 1 mM oxidized glutathione and 2 mM reduced glutathione/0.3 M urea) at 22C for 24 h and then refolded in buffer II (50 mM Tris –HCl, pH 8.0/1% glycerol/0.01% Tween 20/5 mM NaCl) at 4C for another 24 h. The refolded protein was concentrated to 1 mg/ml with Centricon (Amicon, Beverly, MA) at 4C. Functional analysis of refolded protein was carried out in 7.5 ul PBS containing 1 mM CaCl2 and 10 ug fibrinogen at 37C for 0, 1, 3, 6, 8, and 14 h, respectively. The protein ratio of fibrinogen to the refolded protein was 100/ 1(w/w). Digestion reactions were stopped by heating the reaction mixtures at 100C for 5 min and the samples were electrophoresed on SDS gels. |
| Unspecified |
| None |
| Glycerol,tween 20 |
| 20%-0.01% |
| n/a |
| n/a |
| Diluted protein was refolded in two step. First in buffer I (50 mM Tris –HCl, pH 8.0/0.1 mM EDTA/0.75 M NaCl/0.01% Tween 20/20% glycerol/ 1 mM oxidized glutathione and 2 mM reduced glutathione/0.3 M urea) at 22C for 24 h and then refolded in buffer II (50 mM Tris –HCl, pH 8.0/1% glycerol/0.01% Tween 20/5 mM NaCl) at 4C for another 24 h. |