Refolding Record:
| Protein | |
|---|---|
| Protein Name | Beta galactoside alpha 2,6 sialyltransferase |
| Abbreviated Name | sialyltransferase 1 |
| SCOP Family | Unknown |
| Structure Notes | |
| Organism | Mouse |
| UniProt Accession | Q8K1L1 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Unknown |
| Molecularity | Unknown |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 376 |
| Molecular Weight | 43335.6 |
| Pi | 8.6614 |
| Molecular Weight | 43335.6 |
| Disulphides | 0 |
| Full Sequence |
MGS DYEALTLQAK VFQMPKSQEK VAVGPAPQAV FSNSKQDPKE GVQILSYPRV TAKVKPQPSL QVWDKDSTYS KLNPRLLKIW RNYLNMNKYK VSYKGPGPGV KFSVEALRCH LRDHVNVSMI
EATDFPFNTT EWEGYLPKEN FRTKAGPWHK CAVVSSAGSL KNSQLGREID NHDAVLRFNG APTDNFQQDV GTKTTIRLVN SQLVTTEKRF LKDSLYTEGI LILWDPSVYH ADIPQWYQKP DYNFFETYKS YRRLHPSQPF YILKPQMPWE LWDIIQEISP DLIQPNPPSS GMLGIIIMMT LCDQVDIYEF LPSKRKTDVC
YYHQKFFDSA CTMGAYHPLL FEKNMVKHLN EGTDEDIYLF GKATLSGFRN NRC
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Hamamoto T, Lee Y-C, Kurosawa N, Nakaoka T, Kojima N, Tsuji S (1994) BIO/TECHNOLOGY, 2, 79-84 |
| Project Aim | Functional Studies |
| Fusion | None |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | JM109(DE3) |
| Expression Temp | 37.0 |
| Expression Time | 2h |
| Expression Vector | pET3b |
| Expression Protocol | Cells were grown in 100ml LB medium (with 100microg/ml ampicillin) at 37degC. When OD600 reached 0.2-0.4, expression was induced by the addition of 2mM IPTG. After 2h further incubation, the cells were harvested and then suspended in 10ml of 20mM TrisHCl 8.0. |
| Method of Induction | IPTG |
| Cell Density at Induction | OD 600 = 0.2-0.4 |
| Cell Disruption Method | Chemical |
| Lytic Agent | Lysozyme |
| Pre-Refolding Purification | None |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution |
| Wash Buffer | n/a |
| Solubilization Buffer | 8M urea, 0.3M NaCl, 20mM TrisHCl pH 7.4 |
| Refolding Buffer | 0.5M NaCl, 10mM lactose, 0.5mM EDTA, 20mM MOPS-NaOH, pH 7.0, |
| Pre-Refolding Purification | None |
| Tag Cleaved | yes |
| Refolding pH | 7.5 |
| Refolding Temperature | 25.0 |
| Protein Concentration | |
| Refolding Time | 108h |
| Redox Agent | None |
| Redox Agent Concentration | n/a |
| Refolding Protocol | The cells were treated with 0.1mg/ml lysozyme and 0.01mg/ml DNaseI for 30min, and Triton X-100 was added to a final concentration of 1%. The mixture was centrifuged (12000g, 15min, 4degC) and the precipitate was suspended in 3mL of 10mM TrisHCl pH 7.4. To 0.5ml of the resuspended precipitate, was added 0.48g solid urea, 60 microL 5M NaCl, 20microL 1M TrisHCl, pH 7.4 and water to a final volume of 1ml. The mixture was incubated for 30min at 10degC, followed by centrifugation (15min, 12000g). 0.1mL aliquots of unfolded protein were then diluted each with 1.9ml of refolding buffer containing 2M urea (final protein concentration approximately 0.02mg/ml). The solution was left at 4degC for 12h, then diluted again with an equal volume of refolding buffer without urea, and left at 4degC for a further 48h. The protein was then assayed for activity, before being dialyzed against refolding buffer at 4degC for 48h. |
| Refolding Assay | Enzyme activity |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | |
| Refolding Yield | 0.1U/100ml culture |
| Purity | |
| Notes | |