Refolding Record:

Protein See all refolding records for this protein...
Protein Name	Cyanovirin-N
Abbreviated Name	CV-N
SCOP Family	Cyanovirin-N
Structure Notes
Organism	Nostoc ellipsosporum (Cyanobacterium)
UniProt Accession	P81180
SCOP Unique ID	n/a
Structure Solved
Class	Beta
Molecularity	Dimer

Construct
Full Length	y
Domain	n/a
Chimera	n/a
Variants	n/a
Chain Length	108
Molecular Weight	11967.2
Pi	6.11747
Molecular Weight	11967.2
Disulphides	2
Full Sequence	MLGKFSQTCYN SAIQGSVLTS TCERTNGGYN TSSIDLNSVI ENVDGSLKWQ PSNFIETCRN TQLAGSSELA AECKTRAQQF VSTKINLDDH IANIDGTLKY EHHHHHH
Notes	n/a

Expression
Report	Colleluori DM, Tien D, Kang F, Pagliei T, Kuss R, McCormick T, Watson K, McFadden K, Chaiken I, Buckheit RW, Romano JW (2005) Protein Expression and Purification, 39, 229-236
Project Aim	Drug Studies
Fusion	C-terminal hexahistidine tag
Protein Expression and Production	Protein recombinantly expressed as and refolded from inclusion bodies.
Expression Host	Escherichia coli
Expression Strain	B834(DE3)
Expression Temp	37.0
Expression Time	2h
Expression Vector	pET30b+
Expression Protocol	For 1L cultures, cells were grown in LB-kanamycin (30microg/ml) until OD600 reached 1.2. Expression was induced with 0.1mM IPTG and incubation continued a further 2h. For large scale fermentation (5L), cells were grown in defined medium with 10g/L glucose as the batch substrate, supplemented with 100mg/L methionine. The cultures were grown until the glucose was completely consumed, followed by nutrient feed initiation with 500ml/L glycerol and 10g/L MgSO4. Cultures were grown at 37degC with pH controlled at pH 6.5 with 15% NH4OH. Cultures were induced when OD600 reached 7.5 and were grown until OD600 was 30. Cells were harvested by centrifugation and pellets were stored at -70degC.
Method of Induction	IPTG
Cell Density at Induction	OD 600 = 1.2/7.5
Cell Disruption Method	Sonication
Lytic Agent	Lysozyme
Pre-Refolding Purification	Washing inclusion body
Solubility	insoluble

Refolding
Refolding Method	Dialysis
Wash Buffer	50mM TrisHCl, 5mM EDTA, 5mM DTT, 2M urea, 2% Triton X-100, pH 7.0
Solubilization Buffer	50mM TrisHCl, 5mM EDTA, 5mM DTT, 6M GdnHCl, pH 7.0
Refolding Buffer	20mM TrisHCl pH 7.0
Pre-Refolding Purification	Washing inclusion body
Tag Cleaved	no
Refolding pH	7.0
Refolding Temperature	4.0
Protein Concentration	n/a
Refolding Time	overnig
Redox Agent	None
Redox Agent Concentration	n/a
Refolding Protocol	Cell pellets were thawed and were suspended in 20ml buffer (100mM TrisHCl, 20mM NaCl, 5mM EDTA, 5mM DTT pH 7.5) per gram of cell paste. A protease inhibitor cocktail was added, followed by 0.1mg/ml lysozyme, and the cells were incubated at ambient temperature for 30min. The cells were then sonicated and centrifuged (20000g, 30min, 4degC). The pellet was then resuspended in 10ml of wash buffer per gram of cells, followed by centrifugation (20000g, 40min, 4degC. This wash step was repeated 4 times and the final pellet was resuspended in 10ml of wash buffer without urea or Triton X-100 per gram of cells, then centrifuged again. The pellet was resuspended in 10ml solubilization buffer per gram of cells, then incubated for 1h at 60degC with gentle mixing. The sample was then centrifuged (20000g, 1h, room temperature), and the supernatant was dizlyed against refolding buffer overnight at 4degC. Following dialysis, the sample was centrifuged (20000g, 30min, 4degC), filtered and the protein was purified using a Q Sepharose column. Selected fractions were then pooled, concentrated and dialysed overnight against distilled, deionized water and lyophilized.
Refolding Assay	Bioactivity,Western Blot,HPLC,SDS-PAGE,Sequence Analysis,Target protein binding
Refolding Chaperones	None
Refolding Additives	None
Additives Concentration	n/a
Refolding Yield	n/a
Purity	99.5%
Notes	n/a

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