Refolding Record:
| Protein | |
|---|---|
| Protein Name | Alpha-1-Antichymotrypsin |
| Abbreviated Name | ACT |
| SCOP Family | Serpins |
| Structure Notes | |
| Organism | Human |
| UniProt Accession | P01011 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Multi-domain proteins (alpha and beta) |
| Molecularity | Monomer |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 400 |
| Molecular Weight | 45265.8 |
| Pi | 5.31969 |
| Molecular Weight | 45265.8 |
| Disulphides | 0 |
| Full Sequence |
HPNSPLD EENLTQENQD RGTHVDLGLA SANVDFAFSL YKQLVLKAPD KNVIFSPLSI STALAFLSLG AHNTTLTEIL
KGLKFNLTET SEAEIHQSFQ HLLRTLNQSS DELQLSMGNA MFVKEQLSLL DRFTEDAKRL YGSEAFATDF QDSAAAKKLI NDYVKNGTRG KITDLIKDLD SQTMMVLVNY IFFKAKWEMP FDPQDTHQSR FYLSKKKWVM VPMMSLHHLT IPYFRDEELS CTVVELKYTG NASALFILPD QDKMEEVEAM LLPETLKRWR
DSLEFREIGE LYLPKFSISR DYNLNDILLQ LGIEEAFTSK ADLSGITGAR NLAVSQVVHK AVLDVFEEGT EASAATAVKI TLLSALVETR TIVRFNRPFL MIIVPTDTQN IFFMSKVTNP KQA
|
| Notes | Mutants K337V and K337A also produced |
| Expression | |
|---|---|
| Report | Im H, Yu MH (2000) Protein Science, 9, 934-941 |
| Project Aim | Undefined |
| Fusion | None |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | BL21 |
| Expression Temp | 40.0 |
| Expression Time | 3h |
| Expression Vector | pEAT8 |
| Expression Protocol | A transformant of Escherichia coli BL21(DE3) was grown on MSZB medium (per liter: 1 g of NH,CI, 3 g of KH,PO, 6 g of Na,HPO, 4 g of glucose, 0.48 g of MgSO,10 g of tryptone, 5 g of NaC1) containing 50 pg/ml ampicillin at 40degC. When A600 reached 0.8, 0.4mM IPTG was added growth was continued for 3h. |
| Method of Induction | IPTG |
| Cell Density at Induction | OD 600 = 0.8 |
| Cell Disruption Method | Sonication |
| Lytic Agent | None |
| Pre-Refolding Purification | None |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution/Dialysis combination |
| Wash Buffer | 50mM Tris-HCl, 50mM NaCl, 1mM EDTA, 1mM beta-mercaptoethanol, 0.5% Triton X-100, pH 8.0 |
| Solubilization Buffer | 50mM Tris-HCl, 1mM beta-mercaptoethanol, 0.5%SDS, pH 8.0 |
| Refolding Buffer | 10mM phosphate, 1mM EDTA, 1mM beta-mercaptoethanol |
| Pre-Refolding Purification | None |
| Tag Cleaved | no tag |
| Refolding pH | 6.5 |
| Refolding Temperature | 25.0 |
| Protein Concentration | |
| Refolding Time | |
| Redox Agent | Beta-mercaptoethanol |
| Redox Agent Concentration | 1mM |
| Refolding Protocol | Recombinant protein was purified from inclusion bodies after refolding and ion-exchange chromatography. The inclusion body pellet was washed twice with wash buffer and was solubilized in solubilization buffer at a protein concentration of 2 mg/ml. The protein was refolded by an initial direct 10-fold dilution of the denatured inclusion bodies, followed by dialysis against 10mM phosphate, 1mM EDTA, 1mM beta-mercaptoethanol, pH 6.5. |
| Refolding Assay | Enzyme activity |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | |
| Refolding Yield | |
| Purity | |
| Notes | |