Refolding Record:
| Protein | |
|---|---|
| Protein Name | Bm95 protein |
| Abbreviated Name | Bm95 |
| SCOP Family | Unknown |
| Structure Notes | |
| Organism | Boophilus microplus |
| UniProt Accession | Q9Y0V1 |
| 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 | 550 |
| Molecular Weight | 61818.0 |
| Pi | 6.47 |
| Molecular Weight | 61818.0 |
| Disulphides | 0 |
| Full Sequence |
ESSICSDFGNEFCRNAECEVVPGAEDDFVCKCPRDNMYFNA
AEKQCEYKDTCKTRECSYGRCVESNPSKGSCVCERSDDLTLQCKIKNDYATDCRNRGGTA
KLRTDGFIGATCDCGEWGAMNKTTRNCVPTTCLRPDLTCKDLCEKNLLQRDSRCCQGWNT
ANCSAAPPADSYCSPGSPKGPDGQCKNACRTKEAGFVCKHGCRSTDKAYECTCPSGSTVA
EDGITCKSISYTVSCTVEQKQTCRPTEDCRVQKGTVLCECPWNQHLVGDTCISDCVDKKC
HEEFMDCGVYMNRQSCYCPWKSRKPGPNVNINERLLNEYYYTVSFTPNISFDSDHCKRYE
DRVLGAIRTSIGKEVFKVEILNCTQDIKARLIAEKPLSKYVLRKLQACEHPIGEWCMMYP
KLLIKKNSATEIEEENLCDSLLKNQEAAYKGQNKCVKVDNLFWFQCADGYTTTYEMTRGR
LRRSVCKAGVSCNENEQLECANKGQICVYENGKANCQCPPDTKPGEIGCIERTTCNPKEI
QECQDKKLECVYKNHKAECKCPDDHECSR
|
| Notes | Details on plasmid were not provided. Sequence refers to that of the Bm95 protein used (residues 19-569) |
| Expression | |
|---|---|
| Report | Boue, O., Farnos, O., Gonzalez, A., Fernandez, R., Acosta, J. A., Valdes, R., Gonzalez, J. L., Guanche, Y., Izquierdo, G., Suarez, M., Dominguez, I., Machado, H., Rodriguez, M., Lleonart, R. (2004) Exp Appl Acarol., 32, 119-128 |
| Project Aim | Recombinant Protein Expression |
| Fusion | None |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Pichia pastoris |
| Expression Strain | BMA-2 |
| Expression Temp | 37.0 |
| Expression Time | not stated |
| Expression Vector | not stated |
| Expression Protocol | cells inoculated into a bioreactor contain supplemented saline medium. Cells were induced and then collected via centrifugation. Harvested cells were disrupted by four passes through a ball mill disruptor in continuous flow using beads of 0.5-0.75mm diameter. |
| Method of Induction | Methanol |
| Cell Density at Induction | OD n/a = n/a |
| Cell Disruption Method | Ball Mill |
| Lytic Agent | None |
| Pre-Refolding Purification | None |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Diafiltration |
| Wash Buffer | n/a |
| Solubilization Buffer | 8M Urea |
| Refolding Buffer | Na2HPO4, NaH2PO4 buffer |
| Pre-Refolding Purification | None |
| Tag Cleaved | no |
| Refolding pH | 8.0 |
| Refolding Temperature | 20.0 |
| Protein Concentration | n/a |
| Refolding Time | n/a |
| Redox Agent | None |
| Redox Agent Concentration | n/a |
| Refolding Protocol | Protein was resuspended and srongly homogenized in solubilization buffer. This suspension was then centrifuged(14,000g) before undergoing a continuous process of diafiltration to remove the excess urea. The recombinant Bm95 was then purfied by acid precipitation at pH 5.0 and the supernatant concentrated and diafiltered in a two-membrane process using refolding buffer. |
| Refolding Assay | Western Blot,SDS-PAGE |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | n/a |
| Refolding Yield | n/a |
| Purity | n/a |
| Notes | n/a |