Refolding Record:
| Protein | |
|---|---|
| Protein Name | Eco29kI restriction endonuclease |
| Abbreviated Name | Eco29kI |
| SCOP Family | Unknown |
| Structure Notes | |
| Organism | Escherichia coli |
| UniProt Accession | Q46944 |
| 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 | 217 |
| Molecular Weight | 24566.6 |
| Pi | 8.36147 |
| Molecular Weight | 24566.6 |
| Disulphides | Unknown |
| Full Sequence |
MHNKKFDRSEHVYRNDSFLELIKDAVRFFSGTPVHSLPPPERFQGAGVYALYYTGHYSLY
DEYSRINRLAYNLPIYVGKAVPAGWRQSRISDHETRAGSELSNRIREHGRNIAKTSNLDL
CDFSCRFVIFEATGSDMISTVEAALIKIYKPLWNTVVDGFGNHTPGAGRFAQAKSDWDVI
HPGREWAEKCTGVHSEPYFIEERIKQYFSKSNFT
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Nikitin D, Mokrishcheva M, Denjmukhametov M, Pertzev A, Zakharova M, Solonin A. (2003) Protein Expression and Purification, 30, 26-31 |
| Project Aim | Structure-Function |
| 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 | 37.0 |
| Expression Time | 4h |
| Expression Vector | pQE-30 |
| Expression Protocol | unknown |
| Method of Induction | Not Stated |
| Cell Density at Induction | OD = |
| Cell Disruption Method | Sonication |
| Lytic Agent | None |
| Pre-Refolding Purification | None |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Column refolding: Size-exclusion chromatography |
| Wash Buffer | 20mM potassium phosphate, 0.4M NaCl, 1mM EDTA, 7mM betamercaptoethanol, 0.5% Triton X-100 pH 7.2 |
| Solubilization Buffer | 20mM potassium phosphate, 0.4M NaCl, 1mM EDTA, 7mM betamercaptoethanol, 0.01% Triton X-100 8M urea, pH 7.2 |
| Refolding Buffer | 20mM potassium phosphate, 0.4M NaCl, 1mM EDTA, 7mM betamercaptoethanol, 0.01% Triton X-100 |
| Pre-Refolding Purification | None |
| Tag Cleaved | no |
| Refolding pH | 7.2 |
| Refolding Temperature | 25.0 |
| Protein Concentration | |
| Refolding Time | |
| Redox Agent | Beta-mercaptoethanol |
| Redox Agent Concentration | n/a |
| Refolding Protocol | The sediment from 0.5 g disrupted 6His-Eco29KI-containing E. coli cells was resuspended in buffer C (20 mM potassium-phosphate buffer, pH 7.2; 0.4 M NaCl; 1 mM EDTA; 7 mM 2-mercapthoethanol; and 0.5% Triton X-100), kept at room temperature for 5 min, and centrifuged in a Beckman J2-21 centrifuge at 18,000 rpm, 4 °C for 1 h. The supernatant was discarded and the pellet was extracted sequentially with buffer C containing 1 and 2 M urea using the same scheme. The pellet was then carefully dissolved in buffer C containing 8 M urea and 0.01% Triton X-100, kept for 5 min at room temperature, and centrifuged in a Beckman J2-21 centrifuge at 18,000 rpm, 4 °C for 1 h. The 8 M urea-soluble protein was applied onto a 40-ml Ultrogel AcA 54 column (Serva, Germany) equilibrated with buffer C containing 0.01% Triton X-100. After gel filtration, the fractions with high 6His-Eco29kI activity were analyzed by 12% Laemmli PAGE [13]. The fractions containing the homogeneous 6His-Eco29kI enzyme were collected, dialyzed against storage buffer, and stored at -20 °C. |
| Refolding Assay | Enzyme activity |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | NULL |
| Refolding Yield | 0.4mg from 0.5 mg of frozen cells |
| Purity | |
| Notes | |