Refolding Record:
| Protein | |
|---|---|
| Protein Name | Somatotropin also known as Growth hormone |
| Abbreviated Name | GH |
| SCOP Family | Long-Chain Cytokines |
| Structure Notes | |
| Organism | Thunnus albacares (Yellowfin tuna) (Neothunnus mac |
| UniProt Accession | P34747 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Alpha |
| Molecularity | Unknown |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 187 |
| Molecular Weight | 21305.2 |
| Pi | 7.85 |
| Molecular Weight | 21305.2 |
| Disulphides | 2 |
| Full Sequence |
QPITDSQRLFSIAVSRIQHLHLLAQRLFSDFESSLQTEEQRQLNKIFLQDFCNSDYIISPIDKHETQRSSVLKLLSISYRLVQSWEFPSRSLSGGSALRNQISPKLSELKTGIHLLIRANQDGAEMFADSSALQLAPYGNYYQSLGADESLRRSYELLACFKKDMHKVETYLTVAKCRLSPEANCTL
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Hsih MH, Kuo JC, Tsai HJ. (1997) Appl microbiol biotechnol, 48, 66-72 |
| Project Aim | 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)pLysS |
| Expression Temp | 37.0 |
| Expression Time | 4-24 h |
| Expression Vector | pKLYP |
| Expression Protocol | E. coli KLYP was used to synthesize ®sh rGH. This strain was derived from BL21(DE3)pLysS (Novagen) but contains the plasmid pKLYP (5.2 kb), which has a 0.6-kb insert of cDNA coding for the mature region of yellow®n porgy pre-growth hormone (Tsai et al. 1995). Cells were grown in a superbroth medium (Lech and Brent 1987) with 50 lg/ml ampicillin and 25 lg/ml chloramphenicol at 37 °C. Isopropyl b-D-thiogalacto-pyranoside (IPTG) and lactose served as inducers for induction. Procedures for protein analysis by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) were previously described (Tsai et al. 1995). Approximately 5 lg of cellular proteins, extracted from transformants that were just beginning induction (at time 0), were loaded into the well for protein analysis. The amounts of proteins extracted from transformants induced for 4, 8, 12, 16, 20 and 24 h, and loaded into the wells, varied. In order to keep the same intensity of total protein bands (background) on the gel, from 5-15 lg cellular proteins were loaded into the wells. |
| Method of Induction | IPTG |
| Cell Density at Induction | OD n/a = n/a |
| Cell Disruption Method | Sonication |
| Lytic Agent | None |
| Pre-Refolding Purification | not specified |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dialysis |
| Wash Buffer | 1% Triton X-100 containing 50 mM EDTA |
| Solubilization Buffer | 6 M guanidine hydrochloride |
| Refolding Buffer | 1 mM EDTA, 50 mM NaCl, 100 mM L-Arg, 2 mM GSSG and 2 mM GSH pH 10 |
| Pre-Refolding Purification | not specified |
| Tag Cleaved | no tag |
| Refolding pH | 10.0 |
| Refolding Temperature | 25.0 |
| Protein Concentration | n/a |
| Refolding Time | n/a |
| Redox Agent | GSH/GSSG |
| Redox Agent Concentration | 2/2 mM |
| Refolding Protocol | Solubilization of IB and renaturation of rGH with a mixture of L-arginine and glutathione Dialysis procedures for this experiment were as described above except that the dialysis bu􏰀er contained various combinations of L-arginine (Arg), oxidized glutathione (GSSG) and glutathione (GSH). There were eight experimental groups: (1) 100 mM Arg alone, (2) 300 mM Arg alone, (3) 1 mM GSSG and 1 mM GSH, (4) 2 mM GSSG and 2 mM GSH, (5) 100 mM Arg, 1 mM GSSG and 1 mM GSH, (6) 100 mM Arg, 2 mM GSSG and 2 mM GSH, (7) 300 mM Arg, 1 mM GSSG and 1 mM GSH, and (8) 300 mM Arg, 2 mM GSSG and 2 mM GSH. No Arg or glutathione was added to the control group. |
| Refolding Assay | Biological assay |
| Refolding Chaperones | None |
| Refolding Additives | L-Arginine |
| Additives Concentration | 100 mM |
| Refolding Yield | n/a |
| Purity | n/a |
| Notes | n/a |