Refolding Record:
| Protein | |
|---|---|
| Protein Name | Streptokinase |
| Abbreviated Name | SK |
| SCOP Family | Staphylokinase/streptokinase |
| Structure Notes | |
| Organism | Streptococcus equisimilis |
| UniProt Accession | P00779 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Alpha+Beta |
| Molecularity | Unknown |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 414 |
| Molecular Weight | 47286.8 |
| Pi | 5.11 |
| Molecular Weight | 47286.8 |
| Disulphides | Unknown |
| Full Sequence |
IAGPEWLLDRPSVNNSQLVVSVAGTVEGTNQDISLKFFEIDLTSRPAHGGKTEQGLSPKSKPFATDSGAMSHKLEKADLLKAIQEQLIANVHSNDDYFEVIDFASDATITDRNGKVYFADKDGSVTLPTQPVQEFLLSGHVRVRPYKEKPIQNQAKSVDVEYTVQFTPLNPDDDFRPGLKDTKLLKTLAIGDTITSQELLAQAQSILNKNHPGYTIYERDSSIVTHDNDIFRTILPMDQEFTYRVKNREQAYRINKKSGLNEEINNTDLISEKYYVLKKGEKPYDPFDRSHLKLFTIKYVDVDTNELLKSEQLLTASERNLDFRDLYDPRDKAKLLYNNLDAFGIMDYTLTGKVEDNHDDTNRIITVYMGKRPEGENASYHLAYDKDRYTEEEREVYSYLRYTGTPIPDNPNDK
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Cherish Babu PV, Srinivas VK, Krishna Mohan V, Krishna E. (2008) J Chromatography B, 861, 218-226 |
| Project Aim | Identification and Characterization,Over expression & Renaturation |
| Fusion | None |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | BL21(DE3)RIL |
| Expression Temp | 30.0 |
| Expression Time | 4 h |
| Expression Vector | BBIL-SK (PUC-19, derivat) |
| Expression Protocol | Single cryovial of working culture was inoculated into 10 ml of Luria Bertini (LB) medium with 50 μg/ml ampicillin and grown for 12 h at 30 deg. C on an orbital shaker (Gallenkemp, Sanyo-UK) at 250 rpm. The culture was transferred into two 1 l flasks containing 200 ml of high cell density media (HCDM) and grown at 30 deg. C for 12 h on an orbital shaker at 250 rpm. This culture was used to inoculate 19 l fermenter (Bioengineering, Switzerland) with 10 l of HCDM (Media composition per l: 28 g glucose, 4 g (NH4)2HPO4, 13.3 g KH2PO4, 1.7 g citric acid, 0.0141 g EDTA, 1.2 g MgSO4·7H2O, 5 mg Ampicillin). Fermentation was carried out in fed-batch mode, using glucose as the carbon source. Inlet air pressure to the fermenter was set at 0.5 VVM (volume of the air/volume of the media/minute). Stirrer speed was set at 300 rpm and temperature was set at 30 deg. C. Dissolved oxygen (DO2) level was maintained at 100% saturation. After 8 h of cultivation, when the DO2 level fell to 50% or below, the stirrer speed and air flow were increased in steps of 100 rpm and 0.5 VVM, respectively, up to maximum rpm of 700 and 2.0 VVM. As and when the DO2 concentration decreases to 0%, addition of 90% glucose was carried out. The cultivation was carried out for 26 h to attain maximum cell density. The culture samples were collected from the fermenter at intervals of every 4 h for monitoring optical density and packed cell volume (PCV). After 26 h of cultivation, the cells were induced by shifting the temperature to 42 deg. C. All other conditions being the same, the temperature was maintained at 42 deg. C for 4 h. At the end of the fermentation period the broth temperature was cooled to 10 deg. C and broth was harvested. |
| Method of Induction | Temperature Shift |
| Cell Density at Induction | OD 0.1-0.5 = 600 |
| Cell Disruption Method | Bead Beater |
| Lytic Agent | None |
| Pre-Refolding Purification | None |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution |
| Wash Buffer | 20 mM Tris-HCl pH 8.0 containing 0.9% sodium chloride solution |
| Solubilization Buffer | 4 M, 6 M of urea and guanidine hydrochloride |
| Refolding Buffer | 20 mM Tris–HCl pH 8.0 with various additives (see table in notes part) |
| Pre-Refolding Purification | None |
| Tag Cleaved | no tag |
| Refolding pH | 8.0 |
| Refolding Temperature | 4.0 |
| Protein Concentration | n/a |
| Refolding Time | n/a |
| Redox Agent | None |
| Redox Agent Concentration | n/a |
| Refolding Protocol | Refolding by rapid dilution The solubilized proteins have been refolded in 20 mM Tris–HCl pH 8.0 with various additives, to obtain the recombinant SK protein of highest activity. The additives used are given in Table 1. The solubilized solution containing the denatured protein was rapidly diluted (1:250 ratio) using 20 mM Tris–HCl, pH 8.0 with or without any of the additives mentioned in Table 1 using a magnetic stirrer. |
| Refolding Assay | Enzyme activity |
| Refolding Chaperones | None |
| Refolding Additives | L-Arginine,Glycerol,Triton X-100 |
| Additives Concentration | See Table 1 |
| Refolding Yield | n/a |
| Purity | n/a |
| Notes | Table 1. Different additives used in the rapid dilution method for renaturation of SK inclusion bodies S. No. Buffer with/without additive 1 20 mM Tris–HCl (8.0) 2 20 mM Tris–HCl (8.0) with 1 M l-arginine 3 20 mM Tris–HCl (8.0) with 1% Triton-X-100 and 10% glycerol 4 20 mM Tris–HCl (8.0) with 10 mM Glycine and 10 mM EDTA 5 20 mM Tris–HCl (8.0) with 100 mM NaCl and 10% glycerol 6 20 mM Tris–HCl (8.0) with 10% SDS and 10% glycerol |