Refolding Record:
| Protein | |
|---|---|
| Protein Name | Single chain Fv MabA34, specific for human apolipoprotein A-I |
| Abbreviated Name | MabA34 |
| SCOP Family | V set domains (antibody variable domain-like) |
| Structure Notes | |
| Organism | Mouse |
| UniProt Accession | n/a |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Beta |
| Molecularity | Monomer |
| Construct | |
|---|---|
| Full Length | n |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 448 |
| Molecular Weight | 48030.5 |
| Pi | 6.72181 |
| Molecular Weight | 48030.5 |
| Disulphides | Unknown |
| Full Sequence |
DVLMTQTPLS LPVSLGDQAS ISCRSSQSIV HTNGNTYLEW YLQKPGQSPK LLIYKVSNRF SGVPDRFSGS GSGTDFTLKI SRVEAEDLGV YYCFQGSHVP RTFGGGTKLE IKRADAAPTV
SIFPPSSEQL TSGGASVVCF LNNFYPKDIN VKWKIDGSER QNGVLNSWTD QDSKDSTYSM SSTLTLTKDE YERHNSYTCE ATHKTSTSPI VKSFNRNEC GGGGSGGGGSGGGGS EVQLVESGAE LMKPGASVKI SCKATDYRFS SYWIEWVKQR PGHGLEWIGD ILPGSGNTNY NERFKGKATF TADTSSNTAY MQLSSLTSED SAVYYCAIPD YWGQGTTLTV SSAKTTPPSV YPLAPGSAAQ TNSMVTLGCL VKGYFPEPVT VTWNSGSLSS GVHTFPAVLQ SDLYTLSSSV TVPSSPRPSE TVTCNVAHPA SSTKVDKKIV PRDC
|
| Notes | Variable regions light and heavy chains linked, connected by (Gly4Ser)3 linker |
| Expression | |
|---|---|
| Report | Cho W, Sohn U, Kwak J (2000) J Biotechnology, 77, 169-178 |
| Project Aim | Recombinant Protein Expression |
| Fusion | None |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | BLR(DE3) |
| Expression Temp | 37.0 |
| Expression Time | 4h |
| Expression Vector | pET29b+ |
| Expression Protocol | Cells were grown until mid-log growth phase, when expression was induced with 0.5mM IPTG. Cells were grown for a further 4h, then harvested by centrifugation (12000g) and resuspended in 1/20 of the original culture volume in 50mM TrisHCl pH 8.0, 2mM EDTA. |
| Method of Induction | IPTG |
| Cell Density at Induction | OD n/a = n/a |
| Cell Disruption Method | Sonication |
| Lytic Agent | None |
| Pre-Refolding Purification | Washing inclusion body |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution/Dialysis combination |
| Wash Buffer | 50mM TrisHCl pH 8.0, 2mM EDTA, 1% Triton X-100 |
| Solubilization Buffer | 8Murea, 50mM TrisHCl pH 8.0, 50mM KCl |
| Refolding Buffer | 1:50mM TrisHCl pH 8.0, 50mM KCl, 2:10mM TrisHCl pH 8.0, 2mM EDTA, 3:10mM TrisHCl pH 7.4, 2mM EDTA |
| Pre-Refolding Purification | Washing inclusion body |
| Tag Cleaved | no |
| Refolding pH | 8.0 |
| Refolding Temperature | 4.0 |
| Protein Concentration | 50microM |
| Refolding Time | 24h+ |
| Redox Agent | None |
| Redox Agent Concentration | n/a,n/a |
| Refolding Protocol | Cells were disrupted by sonication and centrifuged (12000g, 15min, 4degC), then washed 3 times with wash buffer and stored at -20degC. The inclusion bodies were dissolved in 1/50 of the original volume in solubilization buffer and incubated at room temperature for 2h. The suspension was centrifuged (12000g, 10min, 4degC) and the supernatant was then diluted 1:10 into refolding buffer 1 and stored at 4degC for 24h. The solution was then dialysed against refolding buffer 2, followed by refolding buffer 3, and then filtered to remove insoluble aggregates |
| Refolding Assay | Western Blot,SDS-PAGE,ELISA |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | n/a |
| Refolding Yield | 20mg/L culture |
| Purity | n/a |
| Notes | Urea was a better denaturant than GdnHCl - denaturation in GdnHCl resulted more multimeric species forming upon refolding |