Refolding Record:
| Protein | |
|---|---|
| Protein Name | Outer membrane protein G |
| Abbreviated Name | OmpG |
| SCOP Family | Unknown |
| Structure Notes | |
| Organism | Escherichia coli |
| UniProt Accession | P76045 |
| 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 | 281 |
| Molecular Weight | 32907.7 |
| Pi | 4.37943 |
| Molecular Weight | 32907.7 |
| Disulphides | 0 |
| Full Sequence |
MEERNDWHFN IGAMYEIENV EGYGEDMDGL AEPSVYFNAA NGPWRIALAY YQEGPVDYSA GKRGTWFDRP ELEVHYQFLE NDDFSFGLTG GFRNYGYHYV DEPGKDTANM QRWKIAPDWD VKLTDDLRFN GWLSMYKFAN DLNTTGYADT RVETETGLQY TFNETVALRV NYYLERGFNM DDSRNNGEFS TQEIRAYLPL TLGNHSVTPY TRIGLDRWSN WDWQDDIERE GHDFNRVGLF YGYDFQNGLS VSLEYAFEWQ DHDEGDSDKF HYAGVGVNYS
F
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Conlan S, Zhang Y, Cheley S, Bayley H (2000) Biochemistry, 39, 11845-11854 |
| Project Aim | Structure-Function |
| Fusion | None |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | PC2889E |
| Expression Temp | 37.0 |
| Expression Time | 2h |
| Expression Vector | pT7-SMC |
| Expression Protocol | 400ml LB medium with 50microg/ml ampicillin was inoculated with 5ml overnight culture. Cells were grown until A600 reached 0.7, expression was then induced with 0.5mM IPTG and grown for a further 2h. The cells were harvested by centrifugation. |
| Method of Induction | IPTG |
| Cell Density at Induction | OD 600 = 0.7 |
| Cell Disruption Method | Chemical |
| Lytic Agent | Lysozyme |
| Pre-Refolding Purification | Ion-exchange chromatography |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dialysis |
| Wash Buffer | 1M urea, 1% Triton X-100, 10mM TrisHCl pH 8.0 |
| Solubilization Buffer | 8M urea, 10mM TrisHCl pH 8.0 |
| Refolding Buffer | 1% (w/v) Genapol X-080, 10mM TrisHCl pH 8.0 |
| Pre-Refolding Purification | Ion-exchange chromatography |
| Tag Cleaved | no tag |
| Refolding pH | 8.0 |
| Refolding Temperature | 4.0 |
| Protein Concentration | n/a |
| Refolding Time | n/a |
| Redox Agent | None |
| Redox Agent Concentration | n/a |
| Refolding Protocol | The cells were lysed in 5ml of 200microg/ml lysozyme, 1mM PMSF, 10mm TrisHCl, 1mM EDTA pH 8.0 on ice for 15min. The lysate was centrifuged (60000g) and the pellet was washed once in 10ml wash buffer containing Triton X-100, and once without Triton X-100. The pellet was then dissolved at 4degC in 50ml solubilization buffer. The protein was filitered and loaded onto a DEAE-sepharose column. The protein was eluted with a linear gradient of 0-500mM (150ml) NaCl in solubilization buffer. Selected fractions were pooled and dialyzed against 2L of refolding buffer with one buffer change at 4degC. |
| Refolding Assay | Far-UV Circular Dichroism,Bioactivity,SDS-PAGE,Gel filtration chromatography |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | n/a |
| Refolding Yield | 12.5mg/L |
| Purity | n/a |
| Notes | n/a |