Refold - Phosphorylase Kinase Gamma T

Refolding Record:

Protein See all refolding records for this protein...
Protein Name	Phosphorylase Kinase Gamma T
Abbreviated Name	PhK-gammaT
SCOP Family	Unknown
Structure Notes
Organism	Rat
UniProt Accession	P31325
SCOP Unique ID	n/a
Structure Solved
Class	Unknown
Molecularity	Heterodimer

Construct
Full Length	y
Domain	n/a
Chimera	n/a
Variants	n/a
Chain Length	406
Molecular Weight	46677.7
Pi	6.38
Molecular Weight	46677.7
Disulphides	0
Full Sequence	MTLDVGPEDE LPDWAAAKEF YQKYDPKDII GRGVSSVVRR CVHRATGDEF AVKIMEVSAE RLSLEQLEEV RDATRREMHI LRQVAGHPHI ITLIDSYESS SFMFLVFDLM RKGELFDYLT EKVALSEKET RSIMRSLLEA VNFLHVNNIV HRDLKPENIL LDDNMQIRLS DFGFSCHLEP GEKLRELCGT PGYLAPEILK CSMDETHPGY GKEVDLWACG VILFTLLAGS PPFWHRRQIL MLRMIMEGQY QFSSPEWDDR SNTVKDLIAK LLQVDPNARL TAEQALQHPF FERCKGSQPW NLTPRQRFRV AVWTILAAGR VALSSHRLRP LTKNALLRDP YALRPVRRLI DNCAFRLYGH WVKKGEQQNR AALFQHQPPR PFPIIATDLE GDSSAITEDE VTLVRS
Notes	n/a

Expression
Report	Calalb, M. B., Fox, D. T., Hanks, S. K. (1992) J Biol Chem, 267, 1455-1463
Project Aim	Functional Studies,Recombinant Protein Expression
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	1h
Expression Vector	pT7-7
Expression Protocol	Cells were grown in LB broth under ampicillin selection. Expression was induced in 200ml of exponentially growing cultures and incubated with shaking. After incubation cells were harvested by centrifugation and lyzed by the addition of lysis buffer (20mM TrisCl, pH 8.0, 100mM NaCl, 1mM EDTA) per g cell pellet. Lysozyme was added to a final concentration of 1mg/ml and the suspension was incubated for 15min. Lysate was cooled and centrifuged(13,000g for 10min).
Method of Induction	IPTG
Cell Density at Induction	OD n/a = n/a
Cell Disruption Method	Chemical
Lytic Agent	Lysozyme
Pre-Refolding Purification	Washing inclusion body
Solubility	insoluble

Refolding
Refolding Method	Dilution
Wash Buffer	20mM TrisCl, pH 8.0, 100mM NaCl, 1mM EDTA, 0.5% Triton X-100, 10mM EDTA
Solubilization Buffer	6M guanidinium chloride, 50mM TrisHCl, pH 7.4, 1mM DTT
Refolding Buffer	42mM TrisHCl, pH 8.2, 62mM HEPES, 2.5 mM DTT, 0.1 CaCl2, 0.01mg/ml CaM
Pre-Refolding Purification	Washing inclusion body
Tag Cleaved	no tag
Refolding pH	8.2
Refolding Temperature	4.0
Protein Concentration	n/a
Refolding Time	1h
Redox Agent	DTT
Redox Agent Concentration	2.5mM
Refolding Protocol	Insoluble Pellet was washed three times in washing buffer, and then once in TE Buffer (10mM TrisCl, pH 8.0, 1 mM EDTA) and then resuspended in TE buffer and stored. Inclusion bodies at a concentration of 2mg/ml were then solubilized in solubilization buffer. This solution was then diluted 10 fold with refolding buffer to allow refolding to occur.
Refolding Assay	Enzyme activity,Ligand Binding
Refolding Chaperones	None
Refolding Additives	None
Additives Concentration	n/a
Refolding Yield	n/a
Purity	n/a
Notes	n/a