Refolding Record:
| Protein | |
|---|---|
| Protein Name | Elongation factor G |
| Abbreviated Name | EF-G |
| SCOP Family | Unknown |
| Structure Notes | |
| Organism | Staphylococcus aureus |
| UniProt Accession | P68789 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Unknown |
| Molecularity | Unknown |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 692 |
| Molecular Weight | 76481.2 |
| Pi | 4.79 |
| Molecular Weight | 76481.2 |
| Disulphides | Unknown |
| Full Sequence |
AREFSLEKTRNIGIMAHIDAGKTTTTERILYYTGRIHKIGETHEGASQMDWMEQEQDRGITITSAATTAAWEGHRVNIIDTPGHVDFTVEVERSLRVLDGAVTVLDAQSGVEPQTETVWRQATTYGVPRIVFVNKMDKLGANFEYSVSTLHDRLQANAAPIQLPIGAEDEFEAIIDLVEMKCFKYTNDLGTEIEEIEIPEDHLDRAEEARASLIEAVAETSDELMEKYLGDEEISVSELKEAIRQATTNVEFYPVLCGTAFKNKGVQLMLDAVIDYLPSPLDVKPIIGHRASNPEEEVIAKADDSAEFAALAFKVMTDPYVGKLTFFRVYSGTMTSGSYVKNSTKGKRERVGRLLQMHANSRQEIDTVYSGDIAAAVGLKDTGTGDTLCGEKNDIILESMEFPEPVIHLSVEPKSKADQDKMTQALVKLQEEDPTFHAHTDEETGQVIIGGMGELHLDILVDRMKKEFNVECNVGAPMVSYRETFKSSAQVQGKFSRQSGGRGQYGDVHIEFTPNETGAGFEFENAIVGGVVPREYIPSVEAGLKDAMENGVLAGYPLIDVKAKLYDGSYHDVDSSEMAFKIAASLALKEAAKKCDPVILEPMMKVTIEMPEEYMGDIMGDVTSRRGRVDGMEPRGNAQVVNAYVPLSEMFGYATSLRSNTQGRGTYTMYFDHYAEVPKSIAEDIIKKNKGE
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Li JJ, Venkataramana M, Wang AQ, Sanyal S, Janson JC, Su ZG. (2005) Protein Expression and Purification, 40, 327-335 |
| 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) |
| Expression Temp | 30.0 |
| Expression Time | 4 h |
| Expression Vector | pET30 |
| Expression Protocol | The pET30 plasmid containing S. aureus fusA insert was transformed into E. coli BL21 (DE3) for over-expression of EF-G protein. The bacterial cells containing the recombinant plasmid were grown in LB medium containing 50 μg kanamycin/ml at 30 °C until the cell density reached to OD595 of 0.4–0.5. Then, IPTG (isopropyl-β-d-thiogalactopyranoside) was added at 1 mM (final concentration) for induction of the EF-G expression and the growth was continued for four more hours. The over-expressed protein was found mostly in inclusion body (80%) and the rest was in soluble form (20%). The cells were harvested by centrifugation at 4000 rpm for 35 min at 4 °C, and the cell pellet was shock-frozen in liquid nitrogen and saved at −80 °C for further use. The soluble fraction was purified separately and was used as a reference for the protein refolded from inclusion body. |
| Method of Induction | IPTG |
| Cell Density at Induction | OD 0.4-0.5 = 595 |
| Cell Disruption Method | French Press |
| Lytic Agent | None |
| Pre-Refolding Purification | not specified |
| Solubility | partial |
| Refolding | |
|---|---|
| Refolding Method | Column refolding: hydrophobic interaction chromatography |
| Wash Buffer | 50 mM Tris–HCl, pH 8.5, containing 1 mM EDTA and 1% Triton X-100 |
| Solubilization Buffer | 20 ml of 50 mM Tris–HCl, pH 8.7, containing 6 M guanidine hydrochloride (Gu-HCl), 1 mM EDTA, and 150 mM DTT (dithiothreitol) |
| Refolding Buffer | 50 mM Tris–HCl, pH 8.6, containing 5 mM GSH and 0.5 mM GSSG |
| Pre-Refolding Purification | not specified |
| Tag Cleaved | no tag |
| Refolding pH | 8.6 |
| Refolding Temperature | 0.0 |
| Protein Concentration | n/a |
| Refolding Time | n/a |
| Redox Agent | GSH/GSSG |
| Redox Agent Concentration | 5/0.5 mM |
| Refolding Protocol | Refolding of EF-G by isocratic elution with refolding buffer on immobilized PEG 20 K column A column packed with PEG 20 K coupled Sepharose HP (10 × 1.0 cm ID) was equilibrated in 50 mM Tris–HCl, pH 8.6, containing 5 mM GSH and 0.5 mM GSSG. After injection of 100 μl of 50 mM Tris–HCl, pH 8.7, containing 6 M Gu-HCl and 1 mM EDTA a sample of 100 μl unfolded EF-G (24 mg/ml) was loaded and eluted with the equilibration buffer at a flowrate of 0.5 ml/min. |
| Refolding Assay | RP-HPLC,SEC-HPLC |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | n/a |
| Refolding Yield | 70% |
| Purity | n/a |
| Notes | The refolding temperature is not stated |