Refolding Record:
| Protein | |
|---|---|
| Protein Name | Androgen receptor (AR)-associated coregulator 70 |
| Abbreviated Name | ARA70 |
| SCOP Family | Hormone/Growth Factor |
| Structure Notes | |
| Organism | Human |
| UniProt Accession | Q13772 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Unknown |
| Molecularity | Monomer |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 615 |
| Molecular Weight | 69726.1 |
| Pi | 5.72483 |
| Molecular Weight | 69726.1 |
| Disulphides | >10 |
| Full Sequence |
MNTFQDQSGSSSNREPLLRCSDARRDLELAIGGVLRAEQQIKDNLREVKAQIHSCISRHLECLRSREVWLYEQVDLIYQLKEETLQQQAQQLYSLLGQFNCLTHQLECTQNKDLANQVSVCLERLGSLTLKPEDSTVLLFEADTITLRQTITTFGSLKTIQIPEHLMAHASSANIGPFLEKRGCISMPEQKSASGIVAVPFSEWLLGSKPASGYQAPYIPSTDPQDWLTQKQTLENSQTSSRACNFFNNVGGNLKGLENWLLKSEKSSYQKCNSHSTTSSFSIEMEKVGDQELPDQDEMD
LSDWLVTPQESHKLRKPENGSRETSEKFKLLFQSYNVNDWLVKTDSCTNCQGNQPKGVEIENLGNLKCLNDHLEAKKPLSTPSMVTEDWLVQNHQDPCKVEEVCRANEPCTSFAECVCDENCEKEALYKWLLKKEGKDKNGMPVEPKPEPEKHKDSLNMWLCPRKEVIEQTKAPKAMTPSRIADSFQVIKNSPLSEWLIRPPYKEGSPKEVPGTEDRAGKQKFKSPMNTSWCSFNTADWVLPGKKMGNLSQLSSGEDKWLLRKKAQEVLLNSPLQEEHNFPPDHYGLPAVCDLFACMQLKVDKEKWLYRTPLQM
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Singh VK, Jia Z (2005) Protein Expression and Purification, 39, 283-287 |
| Project Aim | Structural Studies |
| Fusion | C-terminal hexahistidine tag |
| 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 | 4h |
| Expression Vector | pET21b |
| Expression Protocol | Not Stated |
| Method of Induction | Not Stated |
| Cell Density at Induction | OD = |
| Cell Disruption Method | Sonication |
| Lytic Agent | None |
| Pre-Refolding Purification | Metal affinity chromatography |
| Solubility | soluble |
| Refolding | |
|---|---|
| Refolding Method | Dialysis |
| Wash Buffer | 0.1 M NaH2PO4-NaOH, 6 M GdnHCI, 300 mM NaCl and 250 mM imidazole, pH 8.0 |
| Solubilization Buffer | 6 M GdnHCI, 0.1 M NaH2PO4 - NaOH, 2% TritonX-100, 2 mM EDTA, and 20 mM DTT, pH 8.0 |
| Refolding Buffer | 20 mM NaH2PO4-NaOH, pH 7.5 containing 20 mM NaCI, and 2% glycerol |
| Pre-Refolding Purification | Metal affinity chromatography |
| Tag Cleaved | no |
| Refolding pH | 7.5 |
| Refolding Temperature | 4.0 |
| Protein Concentration | 5 mg/ml |
| Refolding Time | |
| Redox Agent | GSH |
| Redox Agent Concentration | n/a,n/a,n/a |
| Refolding Protocol | Refolding and One-step purification of recombinant ARA70 The cell pellet was resuspended, under protein denaturing conditions in Buffer A containing 6 M GdnHCI, 0.1 M NaH2PO4 - NaOH, 2% TritonX-100, 2 mM EDTA, and 20 mM DTT, pH 8.0. The cells were disrupted by sonication on ice at high setting for 3 cycles of 20 s with 5 m gap between each cycle and then subjected to rocking in cold room for 2 h. The lysed cells were then centrifuged at 10,000 g for 15 m at 4°C in refrigerated high speed centrifuge (Sorvall). The supernatant was collected and diluted 8-fold with Buffer B containing 0.1 M NaH2PO4-NaOH, 6 M GdnHCI, and 10 mM imidazole, pH 8.0 and loaded on a column containing 7 ml pre-equilibrated Ni-NTA resin (Qiagen). Resin was washed with 10 bed volumes of buffer B followed by 0.1M NaH2PO4-NaOH containing 400 mM NaCI and 100 mM imidazole, pH 8.0. The bound protein was then eluted with 0.1 M NaH2PO4-NaOH containing 300 mM NaCI, and 250 mM imidazole, pH 8.0. Eluted protein was mixed with 5% glycerol and 0.5 M L-Arginine and incubated for 5 h at room temperature. Oxidized glutathione (1 mM) was added to the mixture and incubated over night at 4°C. Purified ARA70 was refolded by dialyzing against 20 mM NaH2PO4-NaOH, pH 7.5 containing 20 mM NaCl, and 2% glycerol for three days with buffer change twice a day. An aliquot of 0.5 ml protein sample was taken after each buffer change in order to monitor protein degradation using SDS-PAGE and proper refolding of the recombinant ARA70 by CD analysis. |
| Refolding Assay | Far-UV Circular Dichroism |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | NULL |
| Refolding Yield | |
| Purity | 99% |
| Notes | |