Refolding Record:
| Protein | |
|---|---|
| Protein Name | Acetylcholinesterase |
| Abbreviated Name | AChE |
| SCOP Family | Acetylcholinesterase-like |
| Structure Notes | |
| Organism | Rat |
| UniProt Accession | Q505J2 |
| 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 | 614 |
| Molecular Weight | 68196.5 |
| Pi | 5.99 |
| Molecular Weight | 68196.5 |
| Disulphides | Unknown |
| Full Sequence |
MRPPWYPLHTPSLASPLLFLLLSLLGGGARAEGREDPQLLVRVRGGQLRGIRLKAPGGPVSAFLGIPFAEPPVGSRRFMPPEPKRPWSGILDATTFQNVCYQYVDTLYPGFEGTEMWNPNRELSEDCLYLNVWTPYPRPTSPTPVLIWIYGGGFYSGASSLDVYDGRFLAQVEGTVLVSMNYRVGTFGFLALPGSRDAPGNVGLLDQRLALQWVQENIAAFGGDPMSVTLFGESAGAASVGMHILSLPSRSLFHRAVLQSGTPNGPWATVSAGEARRRATLLARLVGCPPGGAGGNDTELISCLRTRPAQDLVDHEWHVLPQESIFRFSFVPVVDGDFLSDTPEALINTGDFQDLQVLVGVVKDEGSYFLVYGVPGFSKDNESLISRAQFLAGVRIGVPQASDLAAEAVVLHYTDWLHPEDPAHLRDAMSAVVGDHNVVCPVAQLAGRLAAQGARVYAYIFEHRASTLTWPLWMGVPHGYEIEFIFGLPLDPSLNYTVEERIFAQRLMKYWTNFARTGDPNDPRDSKSPRWPPYTTAAQQYVSLNLKPLEVRRGLRAQTCAFWNRFLPKLLSATDTLDEAERQWKAEFHRWSSYMVHWKNQFDHYSKQERCSDL
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Heim J, Schmidt-Dannert C, Atomi H, Schmid RD. (1998) Biochim Biophys Acta., 1396, 306-319 |
| Project Aim | Recombinant Protein Expression |
| Fusion | C-terminal hexahistidine tag |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | BL321 |
| Expression Temp | 30.0 |
| Expression Time | 3 h |
| Expression Vector | pT1-Omp |
| Expression Protocol | Expression of AChE in E. coli and in vitro reconstitution For the expression of AChE in E. coli, 600 ml cultures that have reached an OD600 of 1.0–1.2 were shifted from 30°C to 42°C for 3 h. 1 g of the harvested cells was resuspended in 10 ml buffer A (1 mM EDTA, 50 mM Tris-buffer, pH 8.0) and sonicated for 10 min (Branson Sonifier 250). After centrifugation (20 min, 15 000 U/min, 4°C), the obtained pellet was resuspended in 8 ml buffer B (100 mM NaCl, 1 mM EDTA, 50 mM Tris-buffer, pH 8.0) and incubated for 20 min at 25°C in the presence of DNase (25 μg/ml), followed by centrifugation. |
| Method of Induction | Not Stated |
| Cell Density at Induction | OD 1.0-1.2 = 600 |
| Cell Disruption Method | Sonication |
| Lytic Agent | None |
| Pre-Refolding Purification | None |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution |
| Wash Buffer | 100 mM NaCl, 10 mM EDTA, 50 mM Tris-buffer, pH 8.0, 0.5% Triton X-100 |
| Solubilization Buffer | 50 mM Tris-buffer, pH 8.0, 7 M Guanidine/HCl |
| Refolding Buffer | 0.5 M l-arginine, 1 M tetramethylammonium chloride, 0.3 mM GSSG, 0.3% PEG 4000, pH 8.5 |
| Pre-Refolding Purification | None |
| Tag Cleaved | no |
| Refolding pH | 8.5 |
| Refolding Temperature | 4.0 |
| Protein Concentration | 20 ug/ml |
| Refolding Time | 72 h |
| Redox Agent | GSSG |
| Redox Agent Concentration | 0.3 mM |
| Refolding Protocol | After centrifugation (20 min, 15 000 U/min, 4°C), the obtained pellet was resuspended in 8 ml buffer B (100 mM NaCl, 1 mM EDTA, 50 mM Tris-buffer, pH 8.0) and incubated for 20 min at 25°C in the presence of DNase (25 μg/ml), followed by centrifugation. The pellet was first washed with 15 ml buffer C (100 mM NaCl, 10 mM EDTA, 50 mM Tris-buffer, pH 8.0, 0.5% Triton X-100) and then with 15 ml buffer B. The solubilization of the partially purified inclusion bodies was generally carried out with 10 ml solubilization buffer (50 mM Tris-buffer, pH 8.0, 7 M Guanidine/HCl) by stirring for 2 h at 25°C. After centrifugation (30 min, 15 000 U/min, 4°C), the solubilized and denatured AChE was diluted with refolding buffer (0.5 M -arginine, 1 M tetramethylammonium chloride, 0.3 mM GSSG, 0.3% PEG 4000, pH 8.5) to a final protein concentration of 20 μg/ml and incubated for 72 h at 4°C. Next, the solution was dialyzed against 5 mM -arginine, pH 8.5, 1 mM EDTA for 18 h at 25°C. The volume of the solution was reduced to 15 ml by ultrafiltration (Filtron, 10 kDa). |
| Refolding Assay | Inhibitory activity |
| Refolding Chaperones | None |
| Refolding Additives | L-Arginine |
| Additives Concentration | 0.5 M |
| Refolding Yield | n/a |
| Purity | n/a |
| Notes | n/a |