Refolding Record:
| Protein | |
|---|---|
| Protein Name | Replication Protein A 70 |
| Abbreviated Name | RPA 70 |
| SCOP Family | DNA Replication |
| Structure Notes | |
| Organism | Escherichia coli |
| UniProt Accession | P27694 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Multi-domain |
| Molecularity | Unknown |
| Construct | |
|---|---|
| Full Length | n |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 616 |
| Molecular Weight | 68138.2 |
| Pi | 6.92 |
| Molecular Weight | 68138.2 |
| Disulphides | Unknown |
| Full Sequence |
MVGQLSEGAIAAIMQKGDTNIKPILQVINIRPITTGNSPPRYRLLMSDGLNTLSSFMLATQLNPLVEEEQLSSNCVCQIHRFIVNTLKDGRRVVILMELEVLKSAEAVGVKIGNPVPYNEGLGQPQVAPPAPAASPAASSRPQPQNGSSGMGSTVSKAYGASKTFGKAAGPSLSHTSGGTQSKVVPIASLTPYQSKWTICARVTNKSQIRTWSNSRGEGKLFSLELVDESGEIRATAFNEQVDKFFPLIEVNKVYYFSKGTLKIANKQFTAVKNDYEMTFNNETSVMPCEDDHHLPTVQFDFTGIDDLENKSKDSLVDIIGICKSYEDATKITVRSNNREVAKRNIYLMDTSGKVVTATLWGEDADKFDGSRQPVLAIKGARVSDFGGRSLSVLSSSTIIANPDIPEAYKLRGWFDAEGQALDGVSISDLKSGGVGGSNTNWKTLYEVKSENLGQGDKPDYFSSVATVVYLRKENCMYQACPTQDCNKKVIDQQNGLYRCEKCDTEFPNFKYRMILSVNIADFQENQWVTCFQESAEAILGQNAAYLGELKDKNEQAFEEVFQNANFRSFIFRVRVKVETYNDESRIKATVMDVKPVDYREYGRRLVMSIRRSALM
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Chiku Hiroyuki (2006) Analytical Biochemistry, 348, 307-314 |
| Project Aim | Protein refolding |
| Fusion | None |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | BL21(DE3)Star |
| Expression Temp | 37.0 |
| Expression Time | 4-6hrs |
| Expression Vector | pET Vectors (novagen) |
| Expression Protocol | Culture grown in 1L SuperBroth (Qbiogene) for 1.5hrs 37 C and induced with 1 mM IPTG followed by incubation for 4-6 hrs at 37 C. |
| Method of Induction | IPTG |
| Cell Density at Induction | OD n/a = n/a |
| Cell Disruption Method | Sonication |
| Lytic Agent | Lysozyme |
| Pre-Refolding Purification | Washing inclusion body |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Absorbtion/Centrifugation |
| Wash Buffer | 1xPBS, 20mM Beta-mercaptoethanol |
| Solubilization Buffer | 6M GuHCl |
| Refolding Buffer | 50mM HEPES, 0.5M NaCl, 0.5%(w/v) PEG20,000, 20mM Beta-mercaptoethanol, 1%(v/v) TWEEN pH7.5 |
| Pre-Refolding Purification | Washing inclusion body |
| Tag Cleaved | no tag |
| Refolding pH | 7.5 |
| Refolding Temperature | 4.0 |
| Protein Concentration | n/a |
| Refolding Time | 10 hrs |
| Redox Agent | Beta-mercaptoethanol |
| Redox Agent Concentration | 20mM,20mM,20mM,20mM |
| Refolding Protocol | Denature inclusion bodies in 6M GuHCl for several days. The protein was then absorbed onto zeolite and incubated for 30mins. Following incubation the samples were centrifuged 10,000 rpm for 5 seconds. They were then washed with PBS x4 and resuspend in 50mM Hepes (pH 7.5), 0.5M NaCl, 0.5% (w/v) PEG 20,000, 20 mM beta mercaptoethanol and 1% (v/v) Tween 20. The mixture was then allowed to refold at 4 C for 10hrs. |
| Refolding Assay | Bioactivity,Bradford assay |
| Refolding Chaperones | None |
| Refolding Additives | Polyethylene glycol (PEG),1% (v/v) Tween |
| Additives Concentration | n/a |
| Refolding Yield | n/a |
| Purity | n/a |
| Notes | n/a |