Refolding Record:
Protein | |
---|---|
Protein Name | Epidermal Growth Factor |
Abbreviated Name | EGP |
SCOP Family | EGF-type module |
Structure Notes | |
Organism | Human |
UniProt Accession | P01133 |
SCOP Unique ID | n/a |
Structure Solved | |
Class | Small Proteins |
Molecularity | Monomer |
Construct | |
---|---|
Full Length | y |
Domain | n/a |
Chimera | n/a |
Variants | n/a |
Chain Length | 53 |
Molecular Weight | 6222.0 |
Pi | 4.77 |
Molecular Weight | 6222.0 |
Disulphides | 2 |
Full Sequence |
NSDSECPLSHDGYCLHDGVCMYIEALDKYACNCVVGYIGERCQYRDLKWWELR
|
Notes | n/a |
Expression | |
---|---|
Report | Sharma K, Babu PV, Sasidhar P, Srinivas VK, Mohan VK, Krishna E. (2008) Protein Expression and Purification, 60, 7-14 |
Project Aim | Protein refolding |
Fusion | N-terminal TrpE + C-terminal six arginine |
Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
Expression Host | Escherichia coli |
Expression Strain | BL21(DE3)RIL |
Expression Temp | 37.0 |
Expression Time | 12 h |
Expression Vector | pET11b |
Expression Protocol | Fermentation with fed-batch mode was carried out using glucose as the carbon source. Initial air flow was kept at 0.25 VVM with 100% saturation of dissolved oxygen (DO2) and initial stirrer speed was set at 300 rpm. During cultivation, when the DO2 level goes below 50%, the stirrer speed and air flow were increased in steps of 50 rpm and 0.25 VVM, respectively, up to maximum rpm of 800 and air flow of 1 VVM. Fed-batch was run based on the DO2 level and maintained at 40–50%, by balancing the level of oxygen and glucose feeding. The cultivation was carried out up to 12 h in the fed-batch mode. After 12 h of cultivation all other conditions being the same, the cells were induced with 1 mM IPTG for 4 h. The culture samples were collected at regular intervals for monitoring cell density and protein expression. |
Method of Induction | IPTG |
Cell Density at Induction | OD n/a = n/a |
Cell Disruption Method | Bead Beater |
Lytic Agent | Detergents |
Pre-Refolding Purification | None |
Solubility | insoluble |
Refolding | |
---|---|
Refolding Method | Column refolding-Expanded bed adsorption chromatography (EBA) |
Wash Buffer | n/a |
Solubilization Buffer | 8 M Urea with 5 mM DTT |
Refolding Buffer | 25 mM potassium phosphate buffer with 2 M urea |
Pre-Refolding Purification | None |
Tag Cleaved | no |
Refolding pH | 6.5 |
Refolding Temperature | 25.0 |
Protein Concentration | n/a |
Refolding Time | n/a |
Redox Agent | None |
Redox Agent Concentration | n/a |
Refolding Protocol | Solubilization and refolding by expanded-bed adsorption chromatography The cell lysate were solubilized in 8 M Urea with 5 mM DTT (Dithiothreitol) for 6 h at room temperature under constant stirring [12,13]. The protein concentration during solubilization was kept between 1 and 1.3 mg/ml. After 6 h of solubilization, the solubilized protein was refolded using expanded-bed adsorption chromatography (Streamline SP matrix Amersham Pharmacia). The solubilized sample was loaded onto pre-equilibrated column (Streamline 100 column, Pharmacia) with 25 mM potassium phosphate buffer with 2 M urea, pH 6.5. Twenty liters of solubilized sample was loaded onto 2 L of the EBA matrix, at room temperature. Two liters of the streamline SP matrix was used with a bed volume of 28 cm. While loading the sample in the EBA mode, the expansion ratio was maintained as 1:2, with the bed reaching a height of 60 cm. Unbound proteins and other impurities were removed by thorough washing of the column in expanded mode with 25 mM potassium phosphate buffer, pH 6.5. After washing the column in expanded bed mode, the bound protein was eluted in ion-exchange mode. The bound proteins were eluted by linear gradient of 0–500 mM NaCl in 25 mM potassium phosphate buffer, pH 6.5. Positive elutes were collected according to the absorbance of the fractions monitored by the online UV monitor using Millipore chromatography system (K-40, Millipore). The fraction containing eluted protein was analyzed by SDS–PAGE as described by Laemmli [14]. |
Refolding Assay | Bioactivity |
Refolding Chaperones | None |
Refolding Additives | None |
Additives Concentration | n/a |
Refolding Yield | n/a |
Purity | n/a |
Notes | n/a |