Refold - Ribonuclease A

Refolding Record:

Protein See all refolding records for this protein...
Protein Name	Ribonuclease A
Abbreviated Name	RNase A
SCOP Family	Ribonuclease A-like
Structure Notes
Organism	Human
UniProt Accession	P07998
SCOP Unique ID	n/a
Structure Solved
Class	Alpha+Beta
Molecularity	Monomer

Construct
Full Length	n
Domain	n/a
Chimera	n/a
Variants	n/a
Chain Length	140
Molecular Weight	16166.7
Pi	8.72
Molecular Weight	16166.7
Disulphides	4
Full Sequence	KE SRAKKFQRQH MDS GTLVPRGSM EG GKSKNDVIPF MDVYKKSACK TRELLVDIIQ EYPDEIEHTY IPSCVVLMRC AGCCNDEALE CVPTETRNVT MEVLRVKQRV SQHNFQLSFT EHTKCECRPK AEVKAKKERC EKPR
Notes	n/a

Expression
Report	Backer MV, Gaynutdinov TI, Gorshkova II, Crouch RJ, Hu T, Aloise R, Arab M, Przekop K, Backer JM (2003) J Controlled Release, 89, 499-511
Project Aim	Drug Studies
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	2
Expression Vector	pET29a(+)
Expression Protocol	Cells were grown in Circle Grow medium (Q-Biogen), expression was induced with 1mM IPTG when OD600 reached 0.5-0.7. Cells were grown for a further 2h at 37degC then harvested by centrifugation (15min x 5000g).
Method of Induction	IPTG
Cell Density at Induction	OD 600 = 0.5-0.7
Cell Disruption Method	Sonication
Lytic Agent	None
Pre-Refolding Purification	None
Solubility	insoluble

Refolding
Refolding Method	Dialysis
Wash Buffer	BugBuster (Novagen)/CelLytic B (Sigma), HisBind buffer (Novagen)
Solubilization Buffer	20mM TrisHCl, 100mM NaCl, 8M Urea pH 8.0
Refolding Buffer	1:20mM TrisHCl 2M Urea, 1mM glutathione, 0.4mM oxidized glutathione, 0.5M arginine, pH 8.0/2: 20mM TrisHCl pH 8.0
Pre-Refolding Purification	None
Tag Cleaved	no tag
Refolding pH	8.0
Refolding Temperature	25.0
Protein Concentration
Refolding Time
Redox Agent	GSH/GSSG
Redox Agent Concentration	1mM/0.4mM,1mM/0.4mM
Refolding Protocol	The cells were lysed using BugBuster reagent (Novagen) according to the instructions of the manufacturer. The inclusion bodies were were centrifuged and washed with BugBuster reagent, followed by CelLytic B reagent (Sigma) diluted 1:10 with water, and then finally 1x HisBind buffer (Novagen). The inclusion bodies were solubilized by sonication in solubilization buffer. The protein was then refolded by a 2-step dialysis process, firstly for 20h against 20 volumes of refolding buffer 1, then for 24h against 100 volumes of refolding buffer 2.
Refolding Assay	Enzyme activity
Refolding Chaperones	None
Refolding Additives	None,L-Arginine
Additives Concentration	0.5M
Refolding Yield
Purity	>95%
Notes	Hu-VEGF - same protocol as for VEGF, see separate record no.1508 Other constructs also purified: HuS (aa.18-125 or 18-127) - see separate protocols, records no.1507, 7k9h3