Refolding Record:
| Protein | |
|---|---|
| Protein Name | Lysyl-tRNA synthetase |
| Abbreviated Name | LysRS |
| SCOP Family | Anticodon-binding domain |
| Structure Notes | |
| Organism | Escherichia coli |
| UniProt Accession | P0A8N3 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Beta |
| Molecularity | Monomer |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 504 |
| Molecular Weight | 57472.2 |
| Pi | 5.1 |
| Molecular Weight | 57472.2 |
| Disulphides | Unknown |
| Full Sequence |
SEQHAQGADAVVDLNNELKTRREKLANLREQGIAFPNDFRRDHTSDQLHAEFDGKENEELEALNIEVAVAGRMMTRRIMGKASFVTLQDVGGRIQLYVARDDLPEGVYNEQFKKWDLGDILGAKGKLFKTKTGELSIHCTELRLLTKALRPLPDKFHGLQDQEARYRQRYLDLISNDESRNTFKVRSQILSGIRQFMVNRGFMEVETPMMQVIPGGAAARPFITHHNALDLDMYLRIAPELYLKRLVVGGFERVFEINRNFRNEGISVRHNPEFTMMELYMAYADYKDLIELTESLFRTLAQDILGKTEVTYGDVTLDFGKPFEKLTMREAIKKYRPETDMADLDNFDSAKAIAESIGIHVEKSWGLGRIVTEIFEEVAEAHLIQPTFITEYPAEVSPLARRNDVNPEITDRFEFFIGGREIGNGFSELNDAEDQAQRFLDQVAAKDAGDDEAMFYDEDYVTALEHGLPPTAGLGIGIDRMVMLFTNSHTIRDVILFPAMRPVK
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Choi SI, Han KS, Kim CW, Ryu KS, Kim BH, Kim KH, Kim SI, Kang TH, Shin HC, Lim KH, Kim HK, Hyun JM, Seong BL. (2008) PLoS ONE, 16, e2677 |
| Project Aim | Folding |
| Fusion | N terminal hexa His tag + TEV cleavage sequence |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | HMS174(DE3)plysE |
| Expression Temp | 37.0 |
| Expression Time | 3 h |
| Expression Vector | pGE-LysRS |
| Expression Protocol | The protein expression, SDS-PAGE analysis, and solubility measurement were performed as described previously [18]. Each expression vector was transformed into the E. coli expression host, HMS174(DE3)plysE (Novagen). A single colony of transformants was inoculated into 2 ml of LB containing both 50 µg/ml ampicillin and 30 µg/ml chloramphenicol, then diluted into 20 ml of the fresh LB. Cells were cultured till the optical density (OD) reached to 0.5 at 600 nm. Proteins were expressed for 3 h after the addition of 1 mM IPTG. The harvested cells from 10 ml of culture broth were suspended in 0.3 ml of PBS, lysed by sonication. Fifty µl of total lysates was mixed with the same volume of 2 X SDS loading buffer. To separate soluble and pellet fractions, the remaining total lysates were centrifuged at 13,000 rpm for 12 min. The insoluble pellet fractions were resuspended with PBS of the same volume of soluble fractions. Fifty µl of soluble fractions and insoluble pellet fractions were mixed with 50 µl of 2 X SDS loading buffer. After boiling, the samples were loaded and run on SDS-PAGE. The loading amounts of samples were normalized by final cell OD600 nm. The gels were stained with Coomassie brilliant blue R-250. The solubility of proteins of interest was estimated on SDS-PAGE using Bio-1D image analysis software (Vilber Lourmat). To coexpress tRNAs, the RNA expression plasmid (pE-tRNALys or pE-tRNAPhe) and the protein expression plasmid (pAra-LysRS-GNB2L1 or pAra-LysRS(K130A)-GNB2L1) was co-transformed into the expression host HMS174(DE3). After addition of 0.5 mM IPTG to the growing cells at the OD600 nm of 0.5, the cells were cultured for 30 min, and then 0.02% L-arabinose was added to induce the expression of fusion proteins. After 3h culture, the cells were harvested. |
| Method of Induction | IPTG |
| Cell Density at Induction | OD 0.5 = 600 |
| Cell Disruption Method | Sonication |
| Lytic Agent | None |
| Pre-Refolding Purification | Metal affinity chromatography |
| Solubility | partial |
| Refolding | |
|---|---|
| Refolding Method | Dilution |
| Wash Buffer | n/a |
| Solubilization Buffer | 6 M guanidine-HCl, 1 mM DTT, and 20 mM Tris-HCl (pH 7.8) |
| Refolding Buffer | 20 mM Tris-HCl (pH 7.8), 1 mM DTT, 50 mM NaCl, 1 mM MgCl2, and various RNA (2 µM or equivalent to 2 µM E. coli tRNALys) |
| Pre-Refolding Purification | Metal affinity chromatography |
| Tag Cleaved | no |
| Refolding pH | 7.8 |
| Refolding Temperature | 25.0 |
| Protein Concentration | n/a |
| Refolding Time | 1.5 h |
| Redox Agent | DTT |
| Redox Agent Concentration | 1 mM |
| Refolding Protocol | In vitro refolding of LysRS The purified LysRS with 6 consecutive histidine residue at its C-terminus was denatured in 6 M guanidine-HCl, 1 mM DTT, and 20 mM Tris-HCl (pH 7.8), to a final concentration of 1.3 µM for 2 h at 37°C. The denatured proteins were 50 fold diluted into the refolding buffer containing 20 mM Tris-HCl (pH 7.8), 1 mM DTT, 50 mM NaCl, 1 mM MgCl2, and various RNA (2 µM or equivalent to 2 µM E. coli tRNALys) and incubated for 1.5 h at 25°C. The enzyme activity of refolded LysRS was analyzed by aminoacylation assay of LysRS as described previously [25]. The refolding mixture was 10 fold diluted into the aminoacylation assay buffer (total volume of 100 µl) containing 20 mM Tris-HCl (pH 7.8), 150 mM KCl, 2 mM ATP, 0.1 mM EDTA, 7 mM MgCl2, 1 µCi of L-[14C]-lysine, and 3.7 µM tRNALys at 30°C. At different time intervals, 10 µl of reaction mixture was mixed with the same volume of 10% (w/v) ice-cold trichloroacetic acid (Sigma), placed on ice for 10 min. The precipitates were filtered through Whatman No.2 filter paper, and washed once with 95% ethanol, followed by air drying. The bound [14C]-lysine was determined with liquid scintillation counter (Beckman). |
| Refolding Assay | Unspecified |
| Refolding Chaperones | RNA |
| Refolding Additives | None |
| Additives Concentration | n/a |
| Refolding Yield | n/a |
| Purity | n/a |
| Notes | n/a |