Refolding Record:

Protein See all refolding records for this protein...
Protein Name	Glyceraldehyde-3-phosphate dehydrogenase
Abbreviated Name	GAPDH
SCOP Family	Unknown
Structure Notes
Organism	Paracoccidioides brasiliensis
UniProt Accession	Q6A547
SCOP Unique ID	n/a
Structure Solved
Class	Unknown
Molecularity	Unknown

Construct
Full Length	y
Domain	n/a
Chimera	n/a
Variants	n/a
Chain Length	338
Molecular Weight	36470.4
Pi	8.25
Molecular Weight	36470.4
Disulphides	Unknown
Full Sequence	MVVKVGINGFGRIGRIVFRNAVEHDDVEIVAVNDPFIETKYAAYMLKYDSTHGQFKGDIQHSSSNNLTVNNKTIHFYQERDPANIPWGKHGVDYVVESTGVFTTTEKAKAHLSGGAKKVIISAPSADAPMFVMGVNEKSYRPDISVLSNASCTTNCLAPLAKVIHDNFGIAEGLMTTIHSYTATQKTVDGPSHKDWRGGRTAAQNIIPSSTGAAKAVGKVIPALNGKLTGMAMRVPTANVSVVDLTCRTEKPVTYDQIKAAVKAASEGELKGILGYSEDALVSTDLNGDPRSSIFDASAGIALNDRFVKLISWYDNEWGYSRRVLDLIAYIAKVDAGK
Notes	n/a

Expression
Report	Barbosa MS, Báo SN, Andreotti PF, de Faria FP, Felipe MS, dos Santos Feitosa L, Mendes-Giannini MJ, Soares CM. (2006) Infection and Immunity, 74, 382-389
Project Aim	Undefined
Fusion	N-terminal hexahistidine tag
Protein Expression and Production	Protein recombinantly expressed as and refolded from inclusion bodies.
Expression Host	Escherichia coli
Expression Strain	None
Expression Temp	37.0
Expression Time	2
Expression Vector	TOPO-pET-100-GAPDH
Expression Protocol	Heterologous expression of P. brasiliensis GAPDH and recombinant protein purification. Bacteria transformed with the TOPO-pET-100-GAPDH construct were grown in LB medium supplemented with ampicillin (100 μg/ml) at 37°C until the optical density at 600 nm reached 0.6. Synthesis of the recombinant protein was then initiated by adding IPTG (isopropyl-β-d-thiogalactopyranoside) to a final concentration of 0.8 mM to the growing culture. After 2 h, the bacterial cells were harvested by centrifugation at 3,000 × g and lysis was achieved by incubation of the cells with guanidinium lysis buffer (6 M guanidine hydrochloride, 20 mM NaPO4, pH 7.8, 500 mM NaCl) and sonication
Method of Induction	IPTG
Cell Density at Induction	OD 0.6 = 600
Cell Disruption Method	Sonication
Lytic Agent	Chemicals
Pre-Refolding Purification	Ni-NTA agrose chromatography
Solubility	insoluble

Refolding
Refolding Method	Column refolding: Nickel-chelating chromatography
Wash Buffer	50 mM NaPO4, 0.5 M NaCl, 20 mM imidazole
Solubilization Buffer	8 M urea, 20 mM NaPO4, pH 7.8, 500 mM NaCl
Refolding Buffer	40 mM NaPO4, 0.4 M NaCl, 600 mM imidazole
Pre-Refolding Purification	Ni-NTA agrose chromatography
Tag Cleaved	yes
Refolding pH	6.0
Refolding Temperature	25.0
Protein Concentration	n/a
Refolding Time	n/a
Redox Agent	None
Redox Agent Concentration	n/a
Refolding Protocol	The recombinant protein containing the His tag at its N-terminal end was purified on Ni-nitrilotriacetic acid resin (Invitrogen) under hybrid conditions, as follows. The cell lysate was passed through a Ni-nitrilotriacetic acid column equilibrated with denaturing binding buffer (8 M urea, 20 mM NaPO4, pH 7.8, 500 mM NaCl). The column was washed sequentially with denaturing binding buffer, denaturing wash buffer (8 M urea, 20 mM NaPO4, pH 6.0, 500 mM NaCl), and finally native wash buffer (50 mM NaPO4, 0.5 M NaCl, 20 mM imidazole). The bound proteins were eluted with native elution buffer (40 mM NaPO4, 0.4 M NaCl, 600 mM imidazole) to refold the protein, by following the manufacturer\'s instructions. The purity and size of the protein were evaluated by running the purified molecule through 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), followed by Coomassie blue staining.
Refolding Assay	Western Blot,SDS-PAGE
Refolding Chaperones	None
Refolding Additives	None
Additives Concentration	n/a
Refolding Yield	n/a
Purity	n/a
Notes	n/a

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