Refolding Record:

Protein See all refolding records for this protein...
Protein Name	NAD specific glutamate dehydrogenase
Abbreviated Name	NAD-GDH
SCOP Family	Aminoacid dehydrogenase-like, C-terminal domain
Structure Notes
Organism	Halobacterium mediterranei (Haloferax mediterranei
UniProt Accession	Q977U6
SCOP Unique ID	n/a
Structure Solved
Class	Alpha/Beta
Molecularity	Unknown

Construct
Full Length	y
Domain	n/a
Chimera	n/a
Variants	n/a
Chain Length	441
Molecular Weight	47959.2
Pi	4.5
Molecular Weight	47959.2
Disulphides	Unknown
Full Sequence	MQTSEPEGSSPNTAEAASQSSEPKPASESALETARRQLYRAADHLDIDPNVVERLKHPEAVHEVTVPIERDDGSVSVYTGYRAQHDSVRGPYKGGLRYHPGVTRDECVGLRMWMTWKTEVRRDGPIFGGAKGGIAVNPKDLTLDEKERLTRRFTQEIRTIIGPMKDIPAPDMGTDPQTMAWVMDAYSMQEGETVPGVVTGKPPIVGGSEGRDTAPGRSVAIIAREAIDYLSWDIEDTTVAVQGFGSVGAPAARLLDDYGANVVAVSDVNGAIYDPDGLDTHAIPTHEEEPEAVMTHDAPETFSNEELLELDVDVLIPAAVGNVLTAENADDVQANLIVEGANGPTTSAADANFAERGVPVIPDILANAGGVTVSYFEWLQDINRRTWSLERVHDELETEMLNAWSVVRDEYESRDVPWRDAAYIVALSRIAAAHDARGLWP
Notes	n/a

Expression
Report	Díaz S, Pérez-Pomares F, Pire C, Ferrer J, Bonete MJ. (2006) Extremophiles, 10, 105-115
Project Aim	Over expression & Renaturation
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	30.0
Expression Time	1 h
Expression Vector	pET3a
Expression Protocol	The gene encoding GDH was ligated to NdeI/BamHI digested pET3a, as described in Materials and methods. Recombinant genes are transcribed in this vector by the T7 RNA polymerase (Studier et al. 1990; Pire et al. 2001). The gene encoding for T7 RNA polymerase is under the control of the lac promoter and may, therefore, be induced by IPTG. A high expression of recombinant GDH was obtained by addition of IPTG to a culture of E. coli BL21(DE3) transformed with the recombinant plasmid pET3a- HmGDH. The protein samples obtained from the induced cultures were analyzed by SDS-PAGE. The gel, as shown in Fig. 2, displayed a very strong band in the insoluble fraction corresponding in size (subunit molecular mass) to the NAD-GDH from Hfx. mediterranei. This band was absent in the control cells, containing the plasmid pET3a without insert.
Method of Induction	IPTG
Cell Density at Induction	OD n/a = n/a
Cell Disruption Method	Sonication
Lytic Agent	Lysozyme
Pre-Refolding Purification	None
Solubility	partial

Refolding
Refolding Method	Dilution
Wash Buffer	20 mM Tris–HCl buffer, pH 7.4, 2 M NaCl and 1 mM EDTA
Solubilization Buffer	20 mM Tris–HCl buffer pH 8.0, containing 50 mM DTT, 2 mM EDTA and 8 M urea.
Refolding Buffer	100 mM glycine-NaOH pH 8.5, containing 4 M NaCl and 3.25 mM EDTA
Pre-Refolding Purification	None
Tag Cleaved	no tag
Refolding pH	8.5
Refolding Temperature	25.0
Protein Concentration	n/a
Refolding Time	30 h
Redox Agent	None
Redox Agent Concentration	n/a
Refolding Protocol	Solubilization and refolding of recombinant NAD-GDH In order to obtain active enzyme, inclusion bodies were dissolved in 20 mM Tris–HCl buffer pH 8.0, containing 50 mM DTT, 2 mM EDTA and 8 M urea. NAD-GDH activity was restored optimally by 20-fold rapid dilution. The optimum-refolding buffer was 100 mM glycine-NaOH pH 8.5, containing 4 M NaCl and 3.25 mM EDTA. The refolding was also tried with KCl but the results were optimal with NaCl, as shown in Fig. 3. The plot of activity versus time indicates that enzyme refolding was also time dependent, with the maximum of activity about 30 h after the rapid dilution.
Refolding Assay	enzyme activity
Refolding Chaperones	None
Refolding Additives	Glycine
Additives Concentration	100 mM
Refolding Yield	n/a
Purity	n/a
Notes	n/a

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