Refolding Record:
| Protein | |
|---|---|
| Protein Name | Link module from human TSG-6 |
| Abbreviated Name | Llink module |
| SCOP Family | Link domain |
| Structure Notes | |
| Organism | Human |
| UniProt Accession | P98066 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Alpha+Beta |
| Molecularity | Unknown |
| Construct | |
|---|---|
| Full Length | n |
| Domain | Link module |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 98 |
| Molecular Weight | 10926.5 |
| Pi | 9.48 |
| Molecular Weight | 10926.5 |
| Disulphides | 2 |
| Full Sequence |
GVYHREARSGKYKLTYAEAKAVCEFEGGHLATYKQLEAARKIGFHVCAAGWMAKGRVGYPIVKPGPNCGFGKTGIIDYGIRLNRSERWDAYCYNPHAK
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Chang TS, Wan HM, Chen CC, Giridhar R, Wu WT. (2003) biotechnology letters, 25, 1037-2003 |
| Project Aim | Undefined |
| Fusion | C-terminal luciferase |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | DE3 |
| Expression Temp | 37.0 |
| Expression Time | 4 h |
| Expression Vector | pET-Lmluc |
| Expression Protocol | Expression, purification and refolding of inclusion bodies The recombinant E. coli Tuner (DE3) (Novagen) harboring the plasmid pET-Lmluc was cultivated and induced with 1 mM IPTG when the OD600 of the culture reached 0.3. The cells were harvested after 4 h of induction and processed to purify the inclu sion bodies. The purification process used was the same as that of Carrio et al. (2000). |
| Method of Induction | IPTG |
| Cell Density at Induction | OD 0.3 = 600 |
| Cell Disruption Method | Not stated |
| Lytic Agent | None |
| Pre-Refolding Purification | None |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution |
| Wash Buffer | n/a |
| Solubilization Buffer | 10 mM Tris/HCl, pH 9 |
| Refolding Buffer | 10 mM Tris/HCl, pH 9, 200 ug unfolded Lm-luc ml, 1 uM B -mercaptoethanol |
| Pre-Refolding Purification | None |
| Tag Cleaved | no |
| Refolding pH | 9.0 |
| Refolding Temperature | 25.0 |
| Protein Concentration | n/a |
| Refolding Time | 4 h |
| Redox Agent | Beta-mercaptoethanol |
| Redox Agent Concentration | 1 um |
| Refolding Protocol | In order to optimize the conditions of refolding, purified inclusion bodies were refolded at varying pH, protein and B-mercaptoethanol concentrations, and refolding time,according to the method described by Kahmann et al.(1997). The standard conditions for refolding Lm-luc were as follows: 10 mM ris/HCl, pH 9, refolding time of 4 h, 200 µg unfolded Lm-luc ml−1 , and 1 uM B -mercaptoethanol. Small-scale refolding assays (total volume 100 µl) were conducted. In each case,only one parameter was varied while all other conditions were kept constant as in the standard conditions.For pH titrations, 10 mM sodium acetate (pH 6) and 10 mM Tris/HCl (pH 7–10) were used. After refolding, the mixture was centrifuged at 10 000 g and the supernatant was assayed for HA binding capability and luciferase activity. |
| Refolding Assay | Enzyme activity |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | n/a |
| Refolding Yield | n/a |
| Purity | n/a |
| Notes | n/a |