Refolding Record:

Protein See all refolding records for this protein...
Protein Name	Alanine--glyoxylate aminotransferase
Abbreviated Name	AGT
SCOP Family	Cystathionine synthase-like
Structure Notes
Organism	Human
UniProt Accession	P21549
SCOP Unique ID	n/a
Structure Solved
Class	Alpha/Beta
Molecularity	Monomer

Construct
Full Length	y
Domain	n/a
Chimera	n/a
Variants	n/a
Chain Length	436
Molecular Weight	47839.2
Pi	6.34
Molecular Weight	47839.2
Disulphides	0
Full Sequence	MHHHHHHSSGLVPRGSGMKETAAAKFERQHMDSPSLGTDDDDKA MASHKLLVTP PKALLKPLSI PNQLLLGPGP SNLPPRIMAA GGLQMIGSMS KDMYQIMDEI KEGIQYVFQT RNPLTLVISG SGHCALEAAL VNVLEPGDSF LVGANGIWGQ RAVDIGERIG ARVHPMTKDP GGHYTLQEVE EGLAQHKPVL LFLTHGESST GVLQPLDGFG ELCHRYKCLL LVDSVASLGG TPLYMDRQGI DILYSGSQKA LNAPPGTSLI SFSDKAKKKM YSRKTKPFSF YLDIKWLANF WGCDDQPRMY HHTIPVISLY SLRESLALIA EQGLENSWRQ HREAAAYLHG RLQALGLQLF VKDPALRLPT VTTVAVPAGY DWRDIVSYVI DHFDIEIMGG LGPSTGKVLR IGLLGCNATR ENVDRVTEAL RAALQHCPKK KL
Notes	A range of mutants also produced: P11L, I340M, G170R, G41R, G41V, G82E, K209R

Expression
Report	Coulter-Mackie MB, Lian Q, Wong SG (2005) Protein Expression and Purification, 41, 18-26
Project Aim	Functional Studies
Fusion	N-terminal hexahistidine tag and Protein S peptide tag
Protein Expression and Production	Protein recombinantly expressed as and refolded from inclusion bodies.
Expression Host	Escherichia coli
Expression Strain	BL21(DE3)pLysS
Expression Temp	25.0
Expression Time	not stated
Expression Vector	pET30a
Expression Protocol	Cells were grown in LB medium with 30microg/ml kanamycin and 34microg/ml chloramphenicol at 37degC until A600 reached 0.6. Expression was induced at 25degC with 1mM IPTG. Following expression, cells were harvested by centrifugation and resuspended in 1/10 volume of 0.1M potassium phosphate pH 8, 0.1mM pyridoxal phosphate, 1% Triton X-10.
Method of Induction	IPTG
Cell Density at Induction	OD 600 = 0.6
Cell Disruption Method	Freeze/Thaw+Sonication
Lytic Agent	Detergents
Pre-Refolding Purification	Washing inclusion body
Solubility	partial

Refolding
Refolding Method	Dialysis
Wash Buffer	1:50mM Hepes pH 7.0, 500mM NaCl, 1% Triton X-100, 2:50mM Hepes pH 7.0, 100mM NaCl, 1mM EDTA, 0.015% Tween 20, 5% glycerol, 1mM DTT
Solubilization Buffer	2:50mM Hepes pH 7.0, 100mM NaCl, 1mM EDTA, 0.015% Tween 20, 5% glycerol, 1mM DTT, 6M GdnHCl
Refolding Buffer	50mM Hepes pH 7.0, 100mM NaCl, 1mM EDTA, 5% glycerol, 1mM DTT
Pre-Refolding Purification	Washing inclusion body
Tag Cleaved	no
Refolding pH	7.0
Refolding Temperature	25.0
Protein Concentration	0.05-0.1mg/ml
Refolding Time	16h
Redox Agent	DTT
Redox Agent Concentration	1mM,1mM,1mM
Refolding Protocol	Cells were lysed by freezing and thawing, followed by sonication. Samples were centrifuged (20min, 14000g, 4degC). Protein was purified from both the soluble and insoluble fractions. For the insoluble protein, the crude pellets from 50ml culture were washed twice with 10ml wash buffer 1 and once with wash buffer 2. The pellet was then resuspended in 10 volumes (w/v) of buffer A and incubated for 60min on ice with occasional mixing. Following centrifugation at 4degC, the supernatant was diluted 3-fold with refolding buffer, then further diluted with refolding buffer containing 1.5M GdnHCl to a final protein concentration of 0.05-0.1mg/ml. After 30min incubation at room pemperature and then dialysed for 16h against 2 changes of refolding buffer.
Refolding Assay	Enzyme activity,Mass spectrometry
Refolding Chaperones	None
Refolding Additives	Glycerol
Additives Concentration	5%
Refolding Yield	10-15mg/L
Purity	92%
Notes	The protein was refolded in both the presence and absense of pyridoxal phosphate, no difference to protein recovery was observed.

Back to refolding records...