Refolding Record:
| Protein | |
|---|---|
| Protein Name | Glucose dehydrogenase |
| Abbreviated Name | GlcDH |
| SCOP Family | Unknown |
| Structure Notes | |
| Organism | Halobacterium mediterranei (Haloferax mediterranei |
| UniProt Accession | Q977U7 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Unknown |
| Molecularity | Dimer |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 358 |
| Molecular Weight | 39241.9 |
| Pi | 4.52 |
| Molecular Weight | 39241.9 |
| Disulphides | Unknown |
| Full Sequence |
MKAIAVKRGEDRPVVIEKPRPEPESGEALVRTLRVGVDGTDHEVIAGGHGGFPEGEDHLVLGHEAVGVVVDPNDTELEEGDIVVPTVRRPPASGTNEYFERDQPDMAPDGMYFERGIVGAHGYMSEFFTSPEKYLVRIPRSQAELGFLIEPISITEKALEHAYASRSAFDWDPSSAFVLGNGSLGLLTLAMLKVDDKGYENLYCLGRRDRPDPTIDIIEELDATYVDSRQTPVEDVPDVYEQMDFIYEATGFPKHAIQSVQALAPNGVGALLGVPSDWAFEVDAGAFHREMVLHNKALVG
SVNSHVEHFEAATVTFTKLPKWFLEDLVTGVHPLSEFEAAFDDDDTTIKTAIEFSTV
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Ferrer J, Fisher M, Burke J, Sedelnikova SE, Baker PJ, Gilmour DJ, Bonete MJ, Pire C, Esclapez J, Rice DW. (2001) Acta Crystallographica Section D, 57, 1887-1889 |
| Project Aim | Crystallography |
| Fusion | None |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | BL21(DE3) |
| Expression Temp | 0.0 |
| Expression Time | 3 h |
| Expression Vector | pET3 |
| Expression Protocol | The gene coding H. mediterranei GlcDH (ATCC 33500) was cloned in the expression vector pET3a (Novagen) and used to transform the E. coli strain BL21(DE3). The transformed cells were grown at 310 K to an OD600 of 0.5-1.0 in Luria-Bertani (LB) medium containing 50 µg ml-1 ampicillin. Induction of expression was achieved by the addition of IPTG to a final concentration of 0.4 mM and the cells were harvested by centrifugation after 3 h and then frozen. Examination of the cell extract showed that the overexpressed enzyme is present in the insoluble fraction. The cells were resuspended in 20 mM Tris-HCl pH 7.4 containing 2 M NaCl and 1 mM EDTA (buffer A), lysozyme to a final concentration of 100 µg ml-1 and 0.1%(v/v) Triton X-100 and incubated at 303 K for 60 min. Cell disruption was achieved by sonication for 2 × 20 s in a Soniprep-200 sonicator with amplitude 16-18 µm. Debris was removed by centrifugation at 14 000g for 10 min and the insoluble pellet containing the enzyme was washed with buffer A and centrifuged at 10 000g for 5 min. This step was then repeated. The resultant pellet was then resuspended in 20 mM Tris-HCl pH 8.0 containing 8 M urea, 2 mM EDTA and 50 mM DTT and incubated at 310 K for 30 min. |
| Method of Induction | IPTG |
| Cell Density at Induction | OD 0.5-1.0 = 600 |
| Cell Disruption Method | Sonication |
| Lytic Agent | Lysozyme |
| Pre-Refolding Purification | None |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution |
| Wash Buffer | 20 mM Tris-HCl pH 7.4 containing 2 M NaCl and 1 mM EDTA |
| Solubilization Buffer | 20 mM Tris-HCl pH 8.0 containing 8 M urea, 2 mM EDTA and 50 mM DTT |
| Refolding Buffer | 20 mM Tris-HCl pH 7.4 containing 2 M NaCl and 1 mM EDTA,(NH4)2SO4 added to a final concentration of 2.5 M |
| Pre-Refolding Purification | None |
| Tag Cleaved | no tag |
| Refolding pH | 7.4 |
| Refolding Temperature | 0.0 |
| Protein Concentration | n/a |
| Refolding Time | n/a |
| Redox Agent | None |
| Redox Agent Concentration | n/a |
| Refolding Protocol | Cell disruption was achieved by sonication for 2 × 20 s in a Soniprep-200 sonicator with amplitude 16-18 µm. Debris was removed by centrifugation at 14 000g for 10 min and the insoluble pellet containing the enzyme was washed with buffer A and centrifuged at 10 000g for 5 min. This step was then repeated. The resultant pellet was then resuspended in 20 mM Tris-HCl pH 8.0 containing 8 M urea, 2 mM EDTA and 50 mM DTT and incubated at 310 K for 30 min. A 40-fold rapid dilution with buffer A was then performed and (NH4)2SO4 added to a final concentration of 2.5 M. The solution was centrifuged at 27 000g for 30 min. The supernatant was then loaded onto a DEAE-cellulose column equilibrated in 50 mM phosphate buffer pH 6.6 containing 2.5 M (NH4)2SO4 and 1 mM EDTA. The GlcDH protein was eluted with 50 mM phosphate buffer pH 7.4 containing 2 M NaCl and 1 mM EDTA (Pire et al., 2001) and prior to crystallization, protein samples were concentrated to approximately 25 mg ml-1 using a Vivaspin concentrator (30 000 Da MW cutoff; Viva Science). For crystallization of the binary complex of GlcDH with NADP+, the cofactor was added after protein concentration to a final concentration of 1 mM. Crystals were grown using the standard hanging-drop vapour-diffusion technique by mixing small volumes (5-10 µl) of both the free enzyme and its complex with NADP+ with an equal volume of a precipitant solution of sodium citrate over the concentration range 1.4-1.6 M in 100 mM HEPES buffer pH 7.0-8.0 and allowing the mixture to equilibrate by vapour diffusion with reservoirs of precipitant solution at 290 K. After approximately one week, crystals with a hexagonal bipyramid morphology (form I) with maximum dimensions of 0.25 × 0.40 × 0.25 mm were obtained for the free enzyme and crystals with a cubic morphology (form II) with maximum dimensions of 0.6 × 0.6 × 0.4 mm were obtained for the binary complex of the enzyme with NADP+. Form I and II crystals were mounted in X-ray-transparent capillaries. Preliminary data sets were collected by the rotation method with 1° rotations per frame using a MAR345 detector with double-mirror focused Cu K X-rays produced by a Rigaku RU-200 rotating-anode generator. Data were processed and analysed using the HKL suite of programs (Otwinowski & Minor, 1997) and subsequently handled using CCP4 (Collaborative Computational Project, Number 4, 1994). |
| Refolding Assay | Unspecified |
| Refolding Chaperones | None |
| Refolding Additives | (NH4)2SO4 |
| Additives Concentration | 2.5 mM |
| Refolding Yield | n/a |
| Purity | n/a |
| Notes | The refolding temperature is not stated |