Refolding Record:

Protein See all refolding records for this protein...
Protein Name	Growth Hormone
Abbreviated Name	GH
SCOP Family	Long-Chain Cytokines
Structure Notes
Organism	Epinephelus awoara (yellow grouper fish)
UniProt Accession	Q8UW02
SCOP Unique ID	n/a
Structure Solved
Class	Alpha
Molecularity	Monomer

Construct
Full Length	y
Domain	n/a
Chimera	n/a
Variants	n/a
Chain Length	204
Molecular Weight	23060.4
Pi	6.89954
Molecular Weight	23060.4
Disulphides	Unknown
Full Sequence	MDRVVLLLSVVSLGVSSQPITDGQRLFSIAVSRVQHLHLLAQRLFSDFESSLQTEEQRQLNKIFLQDFCNSDYIISPIDKHETQRSSVLKLLSISYRLVESWEFPSRSLSGGSAPRNQIFPKLSELKTGILLLIRANQDGAELFPDSSALQLAPYGNYYQSLGADESLRRTYELLACFKKDMHKVETYLTVAKCRLSPEANCTL
Notes	n/a

Expression
Report	Chang CC, Tsai CT, Chang CY (2002) Protein Engineering, 15, 437-441
Project Aim	Folding,Recombinant Protein Expression
Fusion	None
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	16
Expression Vector	not stated
Expression Protocol	Cells were grown in LB medium until OD600 reached 0.3, then expression was induced with 0.1mM IPTG at 37degC for 16h.
Method of Induction	Not Stated
Cell Density at Induction	OD 600 = 0.3
Cell Disruption Method	Sonication
Lytic Agent	Lysozyme
Pre-Refolding Purification	None
Solubility	insoluble

Refolding
Refolding Method	Dialysis
Wash Buffer	Milli-Q water
Solubilization Buffer	4.5M urea, 10mM Tris, 0.1M 2-mercaptoethanol
Refolding Buffer	1:10mM Tris pH 11.0, 0.1mM 2-mercaptoethanol, 1mM cysteine, 5% glycerol, 2:10mM TrisHCl pH 8.8, 1mM 2-mercaptoethanol
Pre-Refolding Purification	None
Tag Cleaved	no tag
Refolding pH	8.8
Refolding Temperature	4.0
Protein Concentration
Refolding Time	104h
Redox Agent	Cysteine
Redox Agent Concentration	1mM
Refolding Protocol	Cells were grown in LB medium, when OD600 reached 0.3, protein expression was induced with 0.1mM IPTG, cells were incubated for a further 16h at 37degC. The cells were harvested by centrifugation (30min x 4000g), the pellet was then suspended and mixed with ice-cold lysis buffer 1 (50mM TrisHCl, pH 7.8, 200mM NaCl, 0.1mM EDTA, 5% glycerol) followed by the addition of 1/10 volume of lysis buffer 2 (50mM EDAT, 10% Triton X-100). Lysozyme was also added (final concentration 0.01mg/ml) and the mixture was incubated on ice for 3h. The cells were then sonicated and DNAseI added (final concentration 0.3micrograms/ml) and the mixture incubated for 1h at 37degC. The lysate was then centrifuged (30min x4000g) and the insoluble pellet was washed twice with ice-cold Milli-Q water. The entire lysis process was then repeated. The pellet was then dissolved in solubilization buffer and the mixture was adjusted to pH 12 with NaOH to completely dissolve the proteins. The mixture was centrifuged (30min x 4000g) and the pellet removed. The supernatant was then dialyzed against refolding buffer 1 containing 1M urea for 72h at 4degC. The urea was then removed by dialysis against refolding buffer 1 for 24h at 4degC. The mixture was then dialyzed against refolding buffer 2 containing 5% glycerol for 4hr at 4degC, followed by dialysis against refolding buffer 2 for 4h at 4degC.
Refolding Assay	Bioactivity
Refolding Chaperones	None
Refolding Additives	None,Glycerol
Additives Concentration	5%
Refolding Yield
Purity	93%
Notes	- step-wise dialysis refolding method - same protocol for Plecoglossus altivelis growth hormone UniProt ID: Q8UW02_EPIAW Sequence: I TDGQRLFSIA VSRVQHLHLL AQRLFSDFES SLQTEEQRQL NKIFLQDFCN SDYIISPIDK HETQRSSVLK LLSISYRLVE SWEFPSRSLS GGSAPRNQIF PKLSELKTGI LLLIRANQDG AELFPDSSAL QLAPYGNYYQ SLGADESLRR TYELLACFKK DMHKVETYLT VAKCRLSPEA NCTL Number of amino acids: 185 Molecular weight: 21079.0 Theoretical pI: 7.07

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