Refolding Record:
| Protein | |
|---|---|
| Protein Name | Beta-secretase 2 also known as Memapsin-1 |
| Abbreviated Name | BACE2 |
| SCOP Family | Pepsin-like |
| Structure Notes | |
| Organism | Human |
| UniProt Accession | Q9Y5Z0 |
| 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 | 518 |
| Molecular Weight | 56180.4 |
| Pi | 5.04 |
| Molecular Weight | 56180.4 |
| Disulphides | Unknown |
| Full Sequence |
MGALARALLLPLLAQWLLRAAPELAPAPFTLPLRVAAATNRVVAPTPGPGTPAERHADGLALALEPALASPAGAANFLAMVDNLQGDSGRGYYLEMLIGTPPQKLQILVDTGSSNFAVAGTPHSYIDTYFDTERSSTYRSKGFDVTVKYTQGSWTGFVGEDLVTIPKGFNTSFLVNIATIFESENFFLPGIKWNGILGLAYATLAKPSSSLETFFDSLVTQANIPNVFSMQMCGAGLPVAGSGTNGGSLVLGGIEPSLYKGDIWYTPIKEEWYYQIEILKLEIGGQSLNLDCREYNADKAIVDSGTTLLRLPQKVFDAVVEAVARASLIPEFSDGFWTGSQLACWTNSETPWSYFPKISIYLRDENSSRSFRITILPQLYIQPMMGAGLNYECYRFGISPSTNALVIGATVMEGFYVIFDRAQKRVGFAASPCAEIAGAAVSEISGPFSTEDVASNCVPAQSLSEPILWIVSYALMSVCGAILLVLIVLLLLPFRCQRRPRDPEVVNDESSLVRHRWK
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Kim YT, Downs D, Wu S, Dashti A, Pan Y, Zhai P, Wang X, Zhang XC, Lin X. (2002) Eur J Biochem., 269, 5668-5677 |
| Project Aim | Purification & characterization |
| Fusion | N-terminal T7 tag |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | BL21(DE3) |
| Expression Temp | 0.0 |
| Expression Time | 00 |
| Expression Vector | pET11-PB2-T2 |
| Expression Protocol | E. coli BL21 (DE3) cells transformed with the expression vector (pET11-PB2-T1 or pET11-PB2-T2) were grown in Luria–Bertani broth and induced by the addition of isopropyl-b-D-thiogalactopyranoside (final concentration, 1 mM) for inclusion body production. |
| Method of Induction | IPTG |
| Cell Density at Induction | OD n/a = n/a |
| Cell Disruption Method | Not stated |
| Lytic Agent | None |
| Pre-Refolding Purification | not specified |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution |
| Wash Buffer | n/a |
| Solubilization Buffer | 8 M urea, 1 mM glycine, 0.1 mM EDTA, 10 mM b-mercaptoethanol, 10 mM dithiothreitol, 1 mM reduced glutathione, 0.1 mM oxidized glutathione, 20 mM Tris/ |
| Refolding Buffer | 10 vols 20 mM Tris pH 8.0 |
| Pre-Refolding Purification | not specified |
| Tag Cleaved | no |
| Refolding pH | 8.0 |
| Refolding Temperature | 0.0 |
| Protein Concentration | n/a |
| Refolding Time | n/a |
| Redox Agent | None |
| Redox Agent Concentration | n/a |
| Refolding Protocol | The inclusion body was dissolved in 50 mL of a denaturation buffer (8 M urea, 1 mM glycine, 0.1 mM EDTA, 10 mM b-mercaptoethanol, 10 mM dithiothreitol, 1 mM reduced glutathione, 0.1 mM oxidized glutathione, 20 mM Tris/HCl, pH 10.5) to a protein concentration of 1.2 mgÆmL. The denatured proteins were refolded in 10 vols 20 mM Tris base using a rapid dilution method [10,28], followed by adjusting the pH to 8.0. The refolded protein was concentrated by ultrafiltration, and further purified by two steps of chromatography on columns of Sephacryl S-300 (5 · 100 cm, Amersham Pharmacia Biotech) and Resource-Q (1.6 · 3 cm, prepacked, Amersham Pharmacia Biotech). The enzyme fractions obtained from the last column were pooled, concentrated by ultrafiltration, and used for further experiments. |
| Refolding Assay | Activity assay,Circular Dichroism (uv-CD) |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | n/a |
| Refolding Yield | n/a |
| Purity | n/a |
| Notes | n/a |