Refolding Record:
| Protein | |
|---|---|
| Protein Name | Single-chain FV-Asparaginase Fusion protein |
| Abbreviated Name | scFv-ASNase |
| SCOP Family | Glutaminase/Asparaginase |
| Structure Notes | |
| Organism | Escherichia coli O9:H4 (strain HS) |
| UniProt Accession | A8A499 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Alpha/Beta |
| Molecularity | Unknown |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 524 |
| Molecular Weight | 55115.0 |
| Pi | 9.17 |
| Molecular Weight | 55115.0 |
| Disulphides | Unknown |
| Full Sequence |
MAQVQLVQSGAEVKKPGASVKVSCKASGYTFTSYAMHWVRQAPGQRLEWMGWINAGNGNTKYSQKFQGRVTITRDTSASTAYMELSSLRSEDTAVYYCARLTPNKFKSRGHWGQGTLVTVSRGGGGSGGGGSGGGGSSELTQDPAVSVALGQTVRITCQGDSLRSYYASWYQQKPGQAPVLVIYGKNNRPSGIPDRFSGSSSGNTASLTITGAQAEDEADYYCNSRDSSGNHVVFGGGTKLTVLGAAAEQKLISEEDLNGAAMAQVQLVQSGAEVKKPGASVKVSCKASGYTFTSYAMHWVRQAPGQRLEWMGWINAGNGNTKYSQKFQGRVTITRDTSASTAYMELSSLRSEDTAVYYCARLTPNKFKSRGHWGQGTLVTVSRGGGGSGGGGSGGGGSSELTQDPAVSVALGQTVRITCQGDSLRSYYASWYQQKPGQAPVLVIYGKNNRPSGIPDRFSGSSSGNTASLTITGAQAEDEADYYCNSRDSSGNHVVFGGGTKLTVLGAAAEQKLISEEDLNGAA
|
| Notes | The full sequence is the sequence of scFv (BAA82041 from NCBI) and ASNase (A8A499) |
| Expression | |
|---|---|
| Report | Guo L, Wang J, Qian S, Yan X, Chen R, Meng G. (2000) Biotechnol Bioeng, 70, 456-63 |
| Project Aim | Structural Studies |
| 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 | 37.0 |
| Expression Time | 5 h |
| Expression Vector | pET-SLA |
| Expression Protocol | Expression of Fusion Protein in E. coli BL21(DE3) The expression vector pET-SLA was transformed into E. coli BL21 (DE3). An overnight culture (10 mL) of E. coli B21 (DE3) harboring pET-SLA was inoculated into 1 L of LB medium containing 100 􏰅g/mL ampicillin. After incubation at 37°C with shaking to an OD600 of about 0.5, the culture was induced with isopropyl-B􏰂-D-thiogalacto-pyranoside (IPTG) at a final concentration of 1 mM. The cells were grown for an additional 5 h at 37°C, harvested by centrifugation, resuspended in 40 mL of buffer A (50 mM Na2HPO4–NaH2PO4 [pH 8.0]/2 mM ethylene-diamine tetraacetic acid [EDTA]/50 mM NaCl), and stored at −20°C. |
| Method of Induction | IPTG |
| Cell Density at Induction | OD 0.5 = 600 |
| Cell Disruption Method | Sonication |
| Lytic Agent | None |
| Pre-Refolding Purification | None |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution |
| Wash Buffer | buffer A (50 mM Na2HPO4–NaH2PO4 [pH 8.0]/2 mM ethylene-diamine tetraacetic acid [EDTA]/50 mM NaCl),1 M, 2 M, and 3 M urea |
| Solubilization Buffer | buffer A containing 8 M urea and 100 mM B-mercaptoethanol |
| Refolding Buffer | 20 mM Na2HPO4–NaH2PO4 [pH 8.5]/ 50 mM NaCl/2 mM EDTA |
| Pre-Refolding Purification | None |
| Tag Cleaved | no tag |
| Refolding pH | 8.5 |
| Refolding Temperature | 4.0 |
| Protein Concentration | n/a |
| Refolding Time | 6 h |
| Redox Agent | None |
| Redox Agent Concentration | n/a |
| Refolding Protocol | Isolation of Inclusion Bodies and Refolding of Fusion Protein Bacterial cells were lysed by sonication in 10-mL aliquots at 4°C. After centrifugation at 10,000g for 20 min, the pellet was washed with 10 mL of buffer A containing 1 M, 2 M, and 3 M urea, successively. The pellet was finally dissolved in 10 mL of buffer A containing 8 M urea and 100 mM B-mercaptoethanol, and incubated at 4°C for 2 h. The de- natured fusion protein was directly diluted into 100 mL of a refolding buffer (20 mM Na2HPO4–NaH2PO4 [pH 8.5]/50 mM NaCl/2 mM EDTA) and incubated at 4°C for 6 h. The refolding solution was dialyzed at 4°C against 1 L of buffer A containing 0.1 M urea for 12 h. The sample was subse- quently dialyzed for 36 h at 4°C against 4 L of buffer A. After the final dialysis, the aggregated protein was removed by centrifugation at 10,000g for 20 min. The sample was further dialyzed for 36 h against 4 L of 20 mM NH4HCO (pH 8.0) and then concentrated by lyophilization. The sample could be stored at −20°C. |
| Refolding Assay | Western Blot |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | n/a |
| Refolding Yield | n/a |
| Purity | n/a |
| Notes | n/a |