Refolding Record:

Protein See all refolding records for this protein...
Protein Name	RNase E C-terminal Domain
Abbreviated Name	RNase E CTD
SCOP Family	Unknown
Structure Notes
Organism	Escherichia coli
UniProt Accession	P21513
SCOP Unique ID	n/a
Structure Solved
Class	Unknown
Molecularity	Unknown

Construct
Full Length	n
Domain	C-terminal Domain
Chimera	n/a
Variants	n/a
Chain Length	573
Molecular Weight	62429.3
Pi	5.11098
Molecular Weight	62429.3
Disulphides	Unknown
Full Sequence	LSYMLPKLHEEAMALPSEEEFAERKRPEQPALATFAMPDVPPA PTPAEPAAPVVAPAPKAAPATPAAPAQPGLLSRFFGALKALFSGGEETKPTEQPAPKAEA KPERQQDRRKPRQNNRRDRNERRDTRSERTEGSDNREENRRNRRQAQQQTAETRESRQQA EVTEKARTADEQQAPRRERSRRRNDDKRQAQQEAKALNVEEQSVQETEQEERVRPVQPRR KQRQLNQKVRYEQSVAEEAVVAPVVEETVAAEPIVQEAPAPRTELVKVPLPVVAQTAPEQ QEENNADNRDNGGMPRRSRRSPRHLRVSGQRRRRYRDERYPTQSPMPLTVACASPELASG KVWIRYPIVRPQDVQVEEQREQEEVHVQPMVTEVPVAAAIEPVVSAPVVEEVAGVVEAPV QVAEPQPEVVETTHPEVIAAAVTEQPQVITESDVAVAQEVAEQAEPVVEPQEETADIEEV VETAEVVVAEPEVVAQPAAPVVAEVAAEVETVAAVEPEVTVEHNHATAPMTRAPAPEYVP EAPRHSDWQRPTFAFEGKGAAGGHTATHHASAAPARPQPVE
Notes	n/a

Expression
Report	Callaghan AJ, Jukka JP, Ilag LL, Grossmann JG, Chandran V, Kuhnel K, Poljak L, Carpousis AJ, Robinson CV, Symmons MF, Liusi BF (2004) J Mol Biol, 340, 965-979
Project Aim	Functional Studies
Fusion	N-terminal hexahistidine tag
Protein Expression and Production	Protein recombinantly expressed as and refolded from inclusion bodies.
Expression Host	Escherichia coli
Expression Strain	BL21(DE3)
Expression Temp	37.0
Expression Time	3 hours
Expression Vector	pET15b
Expression Protocol	4.1. His-tagged RNase E CTD expression and purification under denaturing conditions The CTD of the rne gene was cloned into a pET15b vector (Novagen). The construct encodes for an N-terminal hexa-histidine tag, followed by a thrombin cleavage site and rne sequence corresponding to the RNase E residues 1–26 followed by 498–1061 (Figure 1B). E. coli strain BL21(DE3) was transformed with the vector for over-expression. Cultures were grown at 37 °C and induced at an absorbance at 600 nm of 0.5–0.6 with 1 mM isopropyl ?--1-thiogalactopyranoside (IPTG). Cells were harvested three hours after induction by centrifugation at 4000g for 20 minutes at 4 °C, washed once with ice-cold phosphate-buffered saline (PBS) supplemented with Complete Block (Roche) protease inhibitors, pelleted again by centrifugation and stored frozen at ?20 °C. Cell pellets were resuspended in ice-cold PBS supplemented with 6.5 M urea (PBS-U) and Complete Block protease inhibitors (Roche), placed in an ice-bath and sonicated for a total of one minute using a low-intensity probe (Misonix). The cellular lysate was clarified by centrifugation at 30,000g for 25 minutes at 4 °C. The cleared lysate was collected and passed through a 0.2 ?m filter and loaded onto a His Trap column (Amersham) equilibrated with PBS-U at 4 °C. The cleared lysate was circulated slowly through the column for 30 minutes. The column was washed with PBS-U containing 20 mM imidazole, and the sample was eluted with PBS-U containing 500 mM imidazole. The washing and elution buffers were supplemented with 500 mM NaCl, Complete Block (Roche) protease inhibitors and 50 mM glycine to scavenge cynate. The eluted CTD was pooled and concentrated in a 30 kDa centrifugal concentrator (Vivascience) at 6000g, and subsequently buffer-exchanged into PBS-U using PD-10 columns (Amersham). The protein was then applied to a HiTrap Q column (Amersham) equilibrated with buffer A (20 mM Tris–HCl (pH 7.6), 6.5 M urea with Complete Block) and eluted with a gradient of 0–100% buffer B (buffer A+1 M NaCl). Fractions containing the CTD were pooled, concentrated to 5 mg/ml and frozen at ?20 °C.
Method of Induction	IPTG
Cell Density at Induction	OD 0.5 = 600
Cell Disruption Method	Sonication
Lytic Agent	None
Pre-Refolding Purification	His Trap and ion exchange
Solubility	soluble

Refolding
Refolding Method	Dilution
Wash Buffer	unknown
Solubilization Buffer	PBS, 6.5 M Urea
Refolding Buffer	20 mM PBS, 250 mM Na2SO4, pH 7.7
Pre-Refolding Purification	His Trap and ion exchange
Tag Cleaved	no
Refolding pH	7.7
Refolding Temperature	4.0
Protein Concentration	n/a
Refolding Time	n/a
Redox Agent	None
Redox Agent Concentration	n/a
Refolding Protocol	4.2. RNase E CTD refolding An aliquot (100 ?l) containing 0.5 mg of RNase E CTD in PBS-U (as described above), was flash diluted 20-fold into 20 mM sodium potassium phosphate buffer (pH 7.7) supplemented with 250 mM Na2SO4. The protein was further dialyzed against a reservoir of the phosphate buffer on ice and finally concentrated to approximately 0.2 mg/ml in a 30 kDa centrifugal concentrator (Vivascience). The final concentration was confirmed by amino acid composition analysis (Protein and Nucleic Acid Chemistry Facility, University of Cambridge).
Refolding Assay	Solution X-ray scattering
Refolding Chaperones	None
Refolding Additives	None
Additives Concentration	n/a
Refolding Yield	n/a
Purity	n/a
Notes	n/a

Back to refolding records...