Refolding Record:
| Protein | |
|---|---|
| Protein Name | Alpha-1-Antitrypsin |
| Abbreviated Name | A1AT |
| SCOP Family | Serpins |
| Structure Notes | |
| Organism | Human |
| UniProt Accession | P01009 |
| 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 | 44324.5 |
| Pi | 5.3684 |
| Molecular Weight | 44324.5 |
| Disulphides | 0 |
| Full Sequence |
EDPQGDAAQKTDTSHHDQDHPTFNKITPNLAEFAFS
LYRQLAHQSNSTNIFFSPVSIATAFAMLSLGTKADTHDEILEGLNFNLTEIPEAQIHEGF
QELLRTLNQPDSQLQLTTGNGLFLSEGLKLVDKFLEDVKKLYHSEAFTVNFGDTEEAKKQ
INDYVEKGTQGKIVDLVKELDRDTVFALVNYIFFKGKWERPFEVKDTEEEDFHVDQVTTV
KVPMMKRLGMFNIQHCKKLSSWVLLMKYLGNATAIFFLPDEGKLQHLENELTHDIITKFL
ENEDRRSASLHLPKLSITGTYDLKSVLGQLGITKVFSNGADLSGVTEEAPLKLSKAVHKA
VLTIDEKGTEAAGAMFLEAIPMSIPPEVKFNKPFVFLMIEQNTKSPLFMGKVVNPTQK
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Bottomley SP and Stone SR (1998) Protein Engineering, 11, 1243-1247 |
| Project Aim | Biophysical Studies |
| Fusion | None |
| 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 | 3h |
| Expression Vector | pTERMAT |
| Expression Protocol | unknown |
| Method of Induction | Not Stated |
| Cell Density at Induction | OD = |
| Cell Disruption Method | Sonication |
| Lytic Agent | Lysozyme |
| Pre-Refolding Purification | None |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution |
| Wash Buffer | 0.5%(v/v) Triton X-100, 50mM Tris pH 8.0, 10mM EDTA, 100mM NaCl |
| Solubilization Buffer | 6M GdnHCl, 100mM DTT, 50mM TrisHCl pH 8.0 |
| Refolding Buffer | 50mM TrisHCl pH 8.0, 5mM DTT |
| Pre-Refolding Purification | None |
| Tag Cleaved | no tag |
| Refolding pH | 8.0 |
| Refolding Temperature | 4.0 |
| Protein Concentration | |
| Refolding Time | overnight |
| Redox Agent | DTT |
| Redox Agent Concentration | n/a |
| Refolding Protocol | 2L of induced E.coli cells were pelleted and resuspended in 30ml of 50mM Tris pH 8.0, 10mM EDTA, 100mM NaCl. The cells were incubated with 100microL of 50mM PMSF and 1ml of lysozyme (10mg/ml) for 30min. The mixture is then sonicated for 6x30sec cycles and centrifuged. The pellet is then resuspended in 30ml of the same buffer and incubated with DNase1 for 15min. The inclusion bodies were then sonicated and then centrifuged again (16000g), then washed three times with 30ml of Wash Buffer with vigourous vortexing in between washes. The inclusion bodies were then dissolved in 10ml solubilization buffer. The reduced and denatured protein was then added by slow drip to 1-1.6L of refolding buffer at a rate of 10microL/min (0.01mg/min). The refolded protein was then loaded on to a Q-sepharose column equilibrated with 50mM TrisHCl pH 8.0, and eluted with a linear gradient of 0-250mM NaCl in equilibration buffer. |
| Refolding Assay | Enzyme activity |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | NULL |
| Refolding Yield | 5-7mg/L cells |
| Purity | |
| Notes | See also Bird et al (2004) Methods, v.32, 169-176 for protocol details Sequence: MDPQGD AAQKTDTSHH DQDHPTFNKI 50 TPNLAEFAFS LYRQLAHQSN STNIFFSPVS IATAFAMLSL GTKADTHDEI 100 LEGLNFNLTE IPEAQIHEGF QELLRTLNQP DSQLQLTTGN GLFLSEGLKL 150 VDKFLEDVKK LYHSEAFTVN FGDTEEAKKQ INDYVEKGTQ GKIVDLVKEL 200 DRDTVFALVN YIFFKGKWER PFEVKDTEEE DFHVDQVTTV KVPMMKRLGM 250 FNIQHCKKLS SWVLLMKYLG NATAIFFLPD EGKLQHLENE LTHDIITKFL 300 ENEDRRSASL HLPKLSITGT YDLKSVLGQL GITKVFSNGA DLSGVTEEAP 350 LKLSKAVHKA VLTIDEKGTE AAGAMFLEAI PMSIPPEVKF NKPFVFLMIE 400 QNTKSPLFMG KVVNPTQK Number of amino acids: 394 Molecular weight: 44326.6 Theoretical pI: 5.43 |