Refolding Record:

Protein See all refolding records for this protein...
Protein Name	Mitogen-activated protein kinase 14 ,p38 alpha
Abbreviated Name	MAP kinase p38 alpha
SCOP Family	Protein kinases, catalytic subunit
Structure Notes
Organism	Human
UniProt Accession	Q16539
SCOP Unique ID	n/a
Structure Solved
Class	Alpha+Beta
Molecularity	Unknown

Construct
Full Length	y
Domain	n/a
Chimera	n/a
Variants	n/a
Chain Length	359
Molecular Weight	41162.1
Pi	5.48
Molecular Weight	41162.1
Disulphides	Unknown
Full Sequence	SQERPTFYRQELNKTIWEVPERYQNLSPVGSGAYGSVCAAFDTKTGLRVAVKKLSRPFQSIIHAKRTYRELRLLKHMKHENVIGLLDVFTPARSLEEFNDVYLVTHLMGADLNNIVKCQKLTDDHVQFLIYQILRGLKYIHSADIIHRDLKPSNLAVNEDCELKILDFGLARHTDDEMTGYVATRWYRAPEIMLNWMHYNQTVDIWSVGCIMAELLTGRTLFPGTDHIDQLKLILRLVGTPGAELLKKISSESARNYIQSLTQMPKMNFANVFIGANPLAVDLLEKMLVLDSDKRITAAQALAHAYFAQYHDPDDEPVADPYDQSFESRDLLIDEWKSLTYDEVISFVPPPLDQEEMES
Notes	n/a

Expression
Report	Cowan RH, Davies RA, Pinheiro TT. (2008) Analytical Biochemistry, 1, 1
Project Aim	Protein refolding
Fusion	None
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	5 h
Expression Vector	n/a
Expression Protocol	Recombinant human p38α was produced as insoluble protein in E. coli strain BL21 (DE3), transformed with an expression plasmid encoding full-length human p38α. Cells were grown to a OD600 of 0.6 at 37 °C, induced for 5 h with 0.4 mM IPTG, and harvested by centrifugation at 8000g for 20 min. Inclusion bodies were prepared using a modification of the protocol described by Georgio and Valax [17]. Briefly, isolated cells were resuspended in ice-cold 50 mM Tris, 150 mM NaCl, 2 mM DTT, pH 9.0, and lysed by sonication. Insoluble material was separated by centrifugation (35,000g for 40 min) and resuspended in the same buffer.
Method of Induction	IPTG
Cell Density at Induction	OD 0.6 = 600
Cell Disruption Method	Sonication
Lytic Agent	None
Pre-Refolding Purification	None
Solubility	insoluble

Refolding
Refolding Method	Dilution
Wash Buffer	2% Triton X-100 and 2 M urea
Solubilization Buffer	50 mM Tris, 150 mM NaCl, 8 M Urea, 10 mM DTT, pH 9.0
Refolding Buffer	various renaturation buffers in a 96-well screen, formatted in four 24-deep-well plates. shown in table 1 page 28
Pre-Refolding Purification	None
Tag Cleaved	no tag
Refolding pH	8.0
Refolding Temperature	4.0
Protein Concentration	n/a
Refolding Time	overnight
Redox Agent	None
Redox Agent Concentration	n/a,n/a,n/a
Refolding Protocol	Refolding of p38α was initiated by rapid dilution of denatured protein into various renaturation buffers in a 96-well screen, formatted in four 24-deep-well plates. A volume of 5 ml of each renaturation buffer was aliquoted into each well. Under rapid agitation at 4 °C, 100 μl of denatured protein solution, at 5 mg/ml in 8 M urea, was added in a single step to each renaturation buffer, for a final protein concentration of 0.1 mg/ml and a final urea concentration of 160 mM. Refolding was allowed to occur under gentle agitation overnight at 4 °C. After refolding, samples identified as containing soluble protein by SDS–PAGE were concentrated 10-fold using Centricon concentrators and then dialyzed against 10 mM Hepes, 150 mM NaCl, pH 7.4. The percentage recovery of refolded protein, as identified by the analytical techniques described below, was calculated by comparing the protein concentration identified by the analytical techniques to the theoretical maximum possible recovery of protein.
Refolding Assay	Far-UV Circular Dichroism,SDS-PAGE
Refolding Chaperones	None
Refolding Additives	None
Additives Concentration	n/a
Refolding Yield	n/a
Purity	n/a
Notes

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