Refolding Record:
| Protein | |
|---|---|
| Protein Name | Mitochondrial Release Factor 1 |
| Abbreviated Name | MRF1 |
| SCOP Family | Unknown |
| Structure Notes | |
| Organism | Saccharomyces cerevisiae |
| UniProt Accession | P30775 |
| 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 | 415 |
| Molecular Weight | 46770.2 |
| Pi | 8.85 |
| Molecular Weight | 46770.2 |
| Disulphides | 0 |
| Full Sequence |
MWLSKFQFPSRSIFKGVFLGHKLPLLVRLTSTTTNSKSNGSIPTQYTELSPLLVKQAEKYEAELKDLDKDLSCGIHFDVNKQKHYAKLSALTDTFIEYKEKLNELKSLQEMIVSDPSLRAEAEQEYAELVPQYETTSSRLVNKLLPPHPFADKPSLLELRPGVGGIEAMIFTQNLLDMYIGYANYRKWKYRIISKNENESGSGIIDAILSIEEAGSYDRLRFEAGVHRVQRIPSTETKGRTHTSTAAVVVLPQIGDESAKSIDAYERTFKPGEIRVDIMRASGKGGQHVNTTDSAVRLTHIPSGIVVSMQDERSQHKNKAKAFTILRARLAEKERLEKEEKERKARKSQVSSTNRSDKIRTYNFPQNRITDHRCGFTLLDLPGVLSGERLDEVIEAMSKYDSTERAKELLESN
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Askarian-Amiri, M. E., Pel, H. J., Guevremont, D., McCaughan, K.K., Pool, E. S., Sumpter, V. G., Tate, W. P. (2000) J Biol Chem, 275, 17241-17248 |
| 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 | BL21 |
| Expression Temp | 37.0 |
| Expression Time | 3 |
| Expression Vector | pET-3a |
| Expression Protocol | 1 L LB media containing antibiotic was inoculated with an overnight culture and induced with 1 mM IT |
| Method of Induction | IPTG |
| Cell Density at Induction | OD 600 = .6 |
| Cell Disruption Method | Sonication |
| Lytic Agent | Lysozyme |
| Pre-Refolding Purification | Washing inclusion body |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution |
| Wash Buffer | 5 mM TrisHCl, pH 7.8, 5 mM KCL and 1 mM DTT |
| Solubilization Buffer | 8 M Urea, 5 mM TrisHCl, pH 7.8, 5mM KCL and 1 mM DTT |
| Refolding Buffer | 5 mM TrisHCl, pH 7.8, 5 mM KCL and 1 mM DTT, 0.5% Tween 20 at RT |
| Pre-Refolding Purification | Washing inclusion body |
| Tag Cleaved | no tag |
| Refolding pH | 7.8 |
| Refolding Temperature | 25.0 |
| Protein Concentration | n/a |
| Refolding Time | n/a |
| Redox Agent | DTT |
| Redox Agent Concentration | 1 mM |
| Refolding Protocol | Pellet was washed twice with washing buffer, once with washing buffer with 1.5% v/v deoxycholate and then with washing buffer again. This pellet was then suspended in solubilising solution. Solubilised protein was refolded by dilution with the washing buffer. Denature protein diluted very slowly 3 ml/h to a concentration of 0.2 mM. Concentration of diluted protein was preformed by Aquacide II and/or ultrifugation. |
| Refolding Assay | Bioactivity |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | n/a |
| Refolding Yield | n/a |
| Purity | n/a |
| Notes | n/a |