Refolding Record:
| Protein | |
|---|---|
| Protein Name | Beta-1,3-glucan recognition protein 2 also known as BetaGRP-2 |
| Abbreviated Name | BGBP-2, BGRP-2 |
| SCOP Family | Unknown |
| Structure Notes | |
| Organism | Manduca sexta (Tobacco hawkmoth) (Tobacco hornworm |
| UniProt Accession | Q8ISB6 |
| 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 | 465 |
| Molecular Weight | 52490.3 |
| Pi | 5.8 |
| Molecular Weight | 52490.3 |
| Disulphides | Unknown |
| Full Sequence |
RGGPYKVPDAKLEAIYPKGLRVSVPDDGYSLFAFHGKLNEEMEGLEAGHWSRDITKAKQGRWIFRDRNAELKLGDKIYFWTYVIKDGLGYRQDNGEWTVTEFVNENGTVVDTSTAPPPVAPAVSEEDQSPGPQWRPCERSLTESLARERVCKGSLVFSEDFDGSSLADLGNWTAEVRFPGEPDYPYNLYTTDGTVGFESGSLVVRPVMTESKYHEGIIYDRLDLERCTGQLGTLECRRESSGGQIVPPVMTAKLATRRSFAFKFGRIDIKAKMPRGDWLIPELNLEPLDNIYGNQRYASGLMRVAFVRGNDVYAKKLYGGPIMSDADPFRSMLLKDKQGLANWNNDYHVYSLLWKPNGLELMVDGEVYGTIDAGDGFYQIAKNNLVSHASQWLKGTVMAPFDEKFFITLGLRVAGIHDFTDGPGKPWENKGTKAMINFWNNRFRWFPTWHDTSLKVDYVRVYAL
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Jiang H, Ma C, Lu ZQ, Kanost MR. (2004) Insect Biochem Mol Biol., 34, 89-100 |
| Project Aim | Undefined |
| Fusion | N-terminal hexahistidine tag |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | M15 |
| Expression Temp | 37.0 |
| Expression Time | 5 h |
| Expression Vector | pQE60 |
| Expression Protocol | A BGRP-2 cDNA fragment encoding amino acid residues 1–464 was amplified by PCR and cloned into the NcoI and PstI sites of the protein expression vector, H6pQE60 (Lee et al., 1994). Correct insertion and sequence of the resulting plasmid, bGRP-2/H6pQE60, were examined to confirm that the recombinant BGRP-2 starts with Met-His-His-His-His-His-His-Ala-Met- Gly-Gln, followed by the mature bGRP-2 sequence. E.coli M15 harboring bGRP-2/H6pQE60 and pREP4 was cultured in LB medium containing 100 lg/ml ampicillin and induced by 1.0 mM isopropyl-1-thio-B-D-galactopyranoside (IPTG) when A600 reached 0.5–0.7. After 5 h induction, the bacteria from 100 ml culture were harvested. |
| Method of Induction | IPTG |
| Cell Density at Induction | OD 0.5-0.7 = 600 |
| Cell Disruption Method | Sonication |
| Lytic Agent | None |
| Pre-Refolding Purification | Metal affinity chromatography |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dialysis |
| Wash Buffer | n/a |
| Solubilization Buffer | 8 M urea |
| Refolding Buffer | 2 M urea, 2 mM reduced glutathione, 0.2 mM oxidized glutathione, 5% glycerol, 200 mM NaCl, 2 mM MgCl2, 50 mM sodium phosphate, pH 6.8 |
| Pre-Refolding Purification | Metal affinity chromatography |
| Tag Cleaved | no |
| Refolding pH | 6.8 |
| Refolding Temperature | 0.0 |
| Protein Concentration | n/a |
| Refolding Time | overnight |
| Redox Agent | GSH/GSSG |
| Redox Agent Concentration | 2/0.2 mM |
| Refolding Protocol | After 5 h induction, the bacteria from 100 ml culture were harvested by centrifugation and lysed by sonication, and the recombinant protein was purified by Ni2+ affinity chromatography in the presence of 8 M urea (Wang et al., 2001). The eluted protein (H6bGRP-2) was renatured by dialysis overnight against two changes of buffer R (2 M urea, 2 mM reduced glutathione, 0.2 mM oxidized glutathione, 5% glycerol, 200 mM NaCl, 2 mM MgCl2, 50 mM sodium phosphate, pH 6.8). |
| Refolding Assay | Binding assay |
| Refolding Chaperones | None |
| Refolding Additives | Glycerol |
| Additives Concentration | 5% |
| Refolding Yield | n/a |
| Purity | n/a |
| Notes | n/a |