Refolding Record:
| Protein | |
|---|---|
| Protein Name | Calcium Channel Beta1b-subunit |
| Abbreviated Name | Beta1b-subunit |
| SCOP Family | Unknown |
| Structure Notes | |
| Organism | Human |
| UniProt Accession | P54283 |
| 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 | 605 |
| Molecular Weight | 66629.3 |
| Pi | 6.22225 |
| Molecular Weight | 66629.3 |
| Disulphides | 0 |
| Full Sequence |
MRGSHHHHHHGMASMTGGQQMGRDLYDDDDK DPSPQGK YSKRKGRFKR SDGSTSSDTT SNSFVRQGSA ESYTSRPSDS DVSLEEDREA LRKEAERQAL AQLEKAKTKP VAFAVRTNVG YNPSPGDEVP VQGVAITFEP KDFLHIKEKY NNDWWIGRLV KEGCEVGFIP SPVKLDSLRL LQEQTLRQNR LSSSKSGDNS SSSLGDVVTG TRRPTPPASA KQKQKSTEHV PPYDVVPSMR PIILVGPSLK GYEVTDMMQK ALFDFLKHRF DGRISITRVT ADISLAKRSV LNNPSKHIII ERSNTRSSLA EVQSEIERIF ELARTLQLVA LDADTINHPA QLSKTSLAPI IVYIKITSPK VLQRLIKSRG KSQSKHLNVQ IAASEKLAQC PPEMFDIILD ENQLEDACEH LAEYLEAYWK ATHPPSRTPP NPLLNRTMAT AALAVSPAPV SNLQGPYLVS GDQPLDRATG EHASVHEYPG ELGQPPGLYP SNHPPGRAGT LWALSRQDTF DADTPGSRNS VYTEPGDSCV DMETDPSEGP GPGDPAGGGT PPARQGSWEE EEDYEEEMTD NRNRGRNKAR YCAEGGGPVL GRNKNELEGW GQGVYIR
|
| Notes | Sequence expressed and refolded: aa.24-597 |
| Expression | |
|---|---|
| Report | Neely A, Garcia-Olivares J, Voswinkel S, Horstkott H, Hidalgo P (2004) J Biol Chem, 279, 21689-21694 |
| Project Aim | Functional Studies |
| Fusion | N-terminal hexahistidine tag |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | BL21 |
| Expression Temp | 25.0 |
| Expression Time | 90min |
| Expression Vector | pRSETB |
| Expression Protocol | Cells were grown at 25degC and protein expression was induced with 0.5mM IPTG for 90 min when OD600 reached 0.7. |
| Method of Induction | IPTG |
| Cell Density at Induction | OD 600 = 0.7 |
| Cell Disruption Method | Sonication |
| Lytic Agent | None |
| Pre-Refolding Purification | None |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Column refolding: Size-exclusion chromatography |
| Wash Buffer | B-PER (Pierce) |
| Solubilization Buffer | 6M GdmHCl, 100mM NaH2PO4, 10mM Tris pH 8.0 |
| Refolding Buffer | 50mM Tris, 300mM NaCl, 1mM EDTA, pH 8.0 |
| Pre-Refolding Purification | None |
| Tag Cleaved | no |
| Refolding pH | 8.0 |
| Refolding Temperature | 12.0 |
| Protein Concentration | |
| Refolding Time | |
| Redox Agent | DTT |
| Redox Agent Concentration | 10mM |
| Refolding Protocol | Cells were harvested by centrifugation and lysed by sonication. Following centrifugation, the pellet was repeatedly washed using mild detergent (B-PER, Pierce). The cells were then dissolved in solubilization buffer and centrifuged for 5min at 10000g. DTT was added to a final concentration of 10mM and the sample was incubated at 95degC for 10 min. The sample was then allowed to cool down and 5ml at a time was loaded onto a Superdex 200 column pre-equilibrated at 12degC with refolding buffer. Selected fractions were pooled and concentrated to 3-6mg/ml. |
| Refolding Assay | Bioactivity,SDS-PAGE |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | |
| Refolding Yield | |
| Purity | |
| Notes | |