Refolding Record:
Protein | |
---|---|
Protein Name | Single chain Fv 1E4 (anti-preS1 of hepatitis B virus) |
Abbreviated Name | scFv 1E4 |
SCOP Family | V set domains (antibody variable domain-like) |
Structure Notes | |
Organism | Human |
UniProt Accession | n/a |
SCOP Unique ID | n/a |
Structure Solved | |
Class | Beta |
Molecularity | Monomer |
Construct | |
---|---|
Full Length | y |
Domain | n/a |
Chimera | n/a |
Variants | n/a |
Chain Length | 285 |
Molecular Weight | 30971.5 |
Pi | 9.02 |
Molecular Weight | 30971.5 |
Disulphides | Unknown |
Full Sequence |
MRGSHHHHHHGMASMTGGQQMGRDLYDDDDK
DIQMTQSPSS LSASVGDRVT ITCRASQGIS NYLAWYQQKP GKVPKLLIYA ASTLQSGVPSRFSGSGSGTD FTLTISSLQP EDVATYYCQKYNSAPLGGGTKVETKGGSSRSSSSGGGGSGGGGQVQLVQSGGGLVKPGGSLRLSCEASGFPFRDAWMTWVRQAPGRGLEWVGRIKKKSDGAATDQKASVKDRFTISRDDSKNMLWLQMNSLKVEDTAVYYCTRSVSSWYEYFYYYYMDLWFDPWGKGTTVTVSS
|
Notes | V(light)-18 residue linker-V(heavy) |
Expression | |
---|---|
Report | Park S-G, Lee J-S, Je E-Y, Kim I-J, Chung J-H, Choi I-H (2000) Biochem Biophys Res Commun., 275, 553-557 |
Project Aim | Drug Studies |
Fusion | N-terminal hexahis + Xpress tag |
Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
Expression Host | Escherichia coli |
Expression Strain | BL21 |
Expression Temp | 22.0 |
Expression Time | 20h |
Expression Vector | pRSET |
Expression Protocol | Cells were grown in 2xTY medium containing 50microg/ml ampicillin for 20h at 22degC. |
Method of Induction | IPTG |
Cell Density at Induction | OD 600 = 0.3-0.6 |
Cell Disruption Method | Sonication |
Lytic Agent | None |
Pre-Refolding Purification | None |
Solubility | insoluble |
Refolding | |
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Refolding Method | Dialysis |
Wash Buffer | n/a |
Solubilization Buffer | 50mM TrisHCl pH 7.6, 6M GdnHCl, 10mM 2-mercaptoethanol |
Refolding Buffer | 100mM TriHCl pH 8.0, 200mM NaCl, 375microM GSSG, 400mM L-arginine |
Pre-Refolding Purification | None |
Tag Cleaved | no |
Refolding pH | 8.0 |
Refolding Temperature | 4.0 |
Protein Concentration | n/a |
Refolding Time | 48h |
Redox Agent | GSSG |
Redox Agent Concentration | 375microM |
Refolding Protocol | Inclusion bodies were obtained following sonication of the cell culture, then denatured by incubation in solubilization buffer at 4degC overnight. The protein was then dialysed against TS buffer (100mM TrisHCl pH 8.0, 200mM NaCl) containing incrementally decreasing concentrations of GdnHCl (3,2,1,0.75,0.5,0.25,0 M). At the 1.0M GdnHCl stage, refolding was encouraged by the addition of GSSG and L-arginine to the solution. Once refolded, the protein was purified via NiNTA chromatography. |
Refolding Assay | Ligand Binding,Western Blot,SDS-PAGE |
Refolding Chaperones | None |
Refolding Additives | L-Arginine |
Additives Concentration | 400mM |
Refolding Yield | 7.38mg/Lculture |
Purity | n/a |
Notes | n/a |