Refolding Record:

Protein See all refolding records for this protein...
Protein Name	Delta crystallin (Argininosuccinate lyase)
Abbreviated Name	Delta crystallin
SCOP Family	Unknown
Structure Notes
Organism	Goose (Anser anser anser)
UniProt Accession	P33110
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	466
Molecular Weight	51372.2
Pi	5.76
Molecular Weight	51372.2
Disulphides	Unknown
Full Sequence	MASEGDKLMGGRFVGSTDPIMQMLSTSMSTEQRLSEVDIQASIAYAKALEKAGILTKTELEKILSGLEKISEEWSKGVFVVTQSDEDIHTANERRLKELIGDIAGKLNTGRSRNEQVVTDLKLFMKNSLSVISTHLLQLIKTLVERAAIEIDVILPGYTHLQKAQPIRWSQFLLSHAVALTRDSERLGEVKRRINVLPLGSGALAGNPLDIDREMLRSELDFASISLNSMDAISERDFVVEFLSVATLLMIHLSKMAEDLIIYSTSEFGFLTLSDAFSTGSSLMPQKKNPDSLELIRSKAGRVFGRLASILMVLKGLPSTYNKDLQEDKEAVFDVVDTLTAVLQVATGVISTLQISKENMEKALTPEMLSTDLALYLVRKGMPFRQAHTASGKAVHLAETKGITINNLTLEDLKSISPLFSSDVSQVFNFVNSVEQYTAMGGTAKSSVTTQIEHLRELMKKQKEQA
Notes	n/a

Expression
Report	Yin FY, Chen YH, Yu CM, Pon YC, Lee HJ. (2007) Biophys J, 93, 1235-1245
Project Aim	Protein refolding
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	27.0
Expression Time	16 h
Expression Vector	pET-8-g delta
Expression Protocol	Protein expression and purification -Crystallin was purified as reported previously (9). Briefly, clones of wild-type or mutant pET-g gene were expressed in Escherichia coli BL21 (DE3) with induction by isopropyl-ß-D-thiogalactopyranoside. After incubation for 16 h at 27°C, cells were harvested by centrifugation and kept frozen at –80°C. Protein concentrations were determined by the protein-dye binding method of Bradford using bovine serum albumin as the standard (24).The frozen cells were resuspended and sonicated in buffer A (50 mM Tris-HCl buffer, pH 7.5).
Method of Induction	IPTG
Cell Density at Induction	OD n/a = n/a
Cell Disruption Method	Sonication
Lytic Agent	None
Pre-Refolding Purification	Ion-exchange chromatography
Solubility	insoluble

Refolding
Refolding Method	Dilution
Wash Buffer	buffer A (50 mM Tris-HCl buffer, pH 7.5)
Solubilization Buffer	various concentrations of GdmCl in 0.1 M Tris-HCl buffer, pH 7.5
Refolding Buffer	buffer A (50 mM Tris-HCl buffer, pH 7.5)
Pre-Refolding Purification	Ion-exchange chromatography
Tag Cleaved	no tag
Refolding pH	7.5
Refolding Temperature	25.0
Protein Concentration	n/a
Refolding Time	n/a
Redox Agent	None
Redox Agent Concentration	n/a,n/a
Refolding Protocol	The supernatants were loaded onto a Q-Sepharose anion exchange column (HiPrep 16/10 Q XL, Amersham Bioscience, Uppsala, Sweden) preequilibrated in buffer A and eluted with a linear gradient of 0–0.3 M NaCl in buffer A. Recombinant goose -crystallin was eluted at 0.12 M NaCl. Ammonium sulfate fractionation was performed on the pooled fractions. The 40 50% (w/v) saturation pellets were dissolved in 5 ml buffer A and loaded onto a S-300 Sephacryl column (26 mm x 85 cm) preequilibrated in buffer A. The highest purity fractions were pooled and concentrated. Porcine lens -crystallin was purified from fresh porcine eye balls. The filtered supernatant sample after homogenization was loaded into a TSK HW55 gel filtration column that has been equilibrated with elution buffer (10 mM Na2HPO4, 2 mM KH2PO4, 3 mM KCl, 5 mM EDTA, and 1.4 mM 2-mercaptoethanol, pH 7.4). The fractions containing -crystallin after SDS-PAGE analysis were pooled and frozen at –80°C.Protein refolding kinetic was monitored by direct dilution of the 5 M GdmCl-denatured -crystallin with buffer A.
Refolding Assay	Fluorescence
Refolding Chaperones	None
Refolding Additives	None
Additives Concentration	n/a
Refolding Yield	n/a
Purity	n/a
Notes	n/a

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