Refolding Record:
| Protein | |
|---|---|
| Protein Name | Alpha-2-macroglobulin |
| Abbreviated Name | Alpha-2-M |
| SCOP Family | Unknown |
| Structure Notes | |
| Organism | Human |
| UniProt Accession | P01023 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Unknown |
| Molecularity | Unknown |
| Construct | |
|---|---|
| Full Length | n |
| Domain | alpha2 M-RBD (res. 1299-1451) |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 152 |
| Molecular Weight | 16924.5 |
| Pi | 6.34 |
| Molecular Weight | 16924.5 |
| Disulphides | Unknown |
| Full Sequence |
LQQVSLPELPGEYSMKVTGEGCVYLQTSLKYNILPEKEEFPFALGVQTLPQTCDEPKAHTSFQISLSVSYTGSRSASNMAIVDVKMVSGFIPLKPTVKMLERSNHVSRTEVSSNHVLIYLDKVSNQTLSLFFTVLQDVPVRDLKPAIVKVYD
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Holtet TL, Nielsen KL, Etzerodt M, Moestrup SK, Gliemann J, Sottrup-Jensen L, Thøgersen HC. (1994) FEBS Letters, 344, 242-246 |
| Project Aim | Expression and charactrization |
| Fusion | ScFX |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | DH1 |
| Expression Temp | 37.0 |
| Expression Time | 3 h |
| Expression Vector | pTTH6ScFX-c~2MRBDv |
| Expression Protocol | Recombinant RBDv protein was produced by expressing the plasmid pTTH6ScFX-c~2MRBDv, which encodes a FXa cleavable fusion protein [21], in E. coli DH1 cells as described [22]. At OD600 : 0.8 exponentially growing cultures at 37°C were infected with bacteriophage A.CE6 at a multiplicity of approx. 5.30 min after infection rifampicin (Sigma) was added and the cultures were grown at 37°C for another 3 h before cells were harvested by centrifugation. Cells were lysed by osmotic shock and sonication and total cellular protein extracted into phenol (adjusted to pH 8.0 with Tris base). Protein was precipitated from the phenol phase by addition of 2.5 vols. of ethanol and isolated by centrifugation. The protein pellet was dissolved in 50 mM Tris-HC1, 6 M guanidinium chloride, 100 mM dithioerythriol, pH 8.0. Following buffer exchange into 50 mM Tris-HC1, 8 M urea, 0.5 M NaC1, 10 mM 2-mercaptoethanol, 2 mM methionine, pH 8.0, by gel filtration on Sephadex G-25 (Pharmacia) the crude protein preparation was applied to a 40 ml Ni activated NTA-Sepharose (Pharmacia) column [23]. The Ni 2+ NTA-Sepharose column was then washed with 50 mM Tris-HCl, 6 M guanidinium chloride, 10 mM 2-mercaptoethanol, 2 mM methionine, pH 8.0 until A 280 of the eluate was stable (approx. 5 column vols.) |
| Method of Induction | Not Stated |
| Cell Density at Induction | OD 0.8 = 600 |
| Cell Disruption Method | Osmotic shock + sonication |
| Lytic Agent | None |
| Pre-Refolding Purification | Metal affinity chromatography |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Column refolding: Nickel-chelating chromatography |
| Wash Buffer | 50 mM Tris-HCl, 6 M guanidinium chloride, 10 mM 2-mercaptoethanol, 2 mM methionine, pH 8.0 |
| Solubilization Buffer | 50 mM Tris-HC1, 6 M guanidinium chloride, 100 mM dithioerythriol, pH 8.0 |
| Refolding Buffer | 50 mM Tris-HC1, 0.5 M NaCI, 10 mM EDTA, pH 8.0 |
| Pre-Refolding Purification | Metal affinity chromatography |
| Tag Cleaved | no |
| Refolding pH | 8.0 |
| Refolding Temperature | 25.0 |
| Protein Concentration | n/a |
| Refolding Time | n/a |
| Redox Agent | None |
| Redox Agent Concentration | n/a |
| Refolding Protocol | Refolding and processing of the alpha2M-RBDv fusion protein. The fusion protein was refolded in vitro, while immobilized on the Ni 2+ NTA-Sepharose column, using an iterative refolding procedure (TLH, ME, and HCT, Patent application no. DK0130/93, DK0139/93; manuscript in preparation). After completion of the iterative folding procedure the fusion protein was eluted from the Ni 2+ NTA-Sepharose column with 50 mM Tris-HC1, 0.5 M NaCI, 10 mM EDTA, pH 8.0. Fusion protein that was aggregated and precipitated on the Ni 2+ NTA- Sepharose column was eluted with 50 mM Tris-HCl, 8 M urea, 0.5 M NaC1, I0 mM 2-mercaptoethanol, 2 mM methionine, pH 8.0. Recombinant RBDv was liberated from the N-terminal fusion tail by cleavage with 1/50 (w/w) of the restriction protease FX a at room temperature for 4 h. After cleavage RBDv was isolated from uncleaved fusion protein, liberated fusion tail and FX a by gel-filtration on Sephadex G-25 in 50 mM Tris-HC1, I0 mM NaC1, pH 8.0, followed by ion-exchange chromatography on Q-Sepharose (Pharmacia). RBDv was eluted with a linear gradient over 10 column vols. from 10 mM to 500 mM NaC1 in 10 mM Tris-HCl, pH 8.0. RBDv eluted at 150 mM NaC1. |
| Refolding Assay | Unspecified |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | n/a |
| Refolding Yield | 45%. |
| Purity | n/a |
| Notes | n/a |