Refolding Record:

Protein See all refolding records for this protein...
Protein Name	Yolk cathepsin
Abbreviated Name	BYC
SCOP Family	Unknown
Structure Notes
Organism	Boophilus microplus (Cattle tick)
UniProt Accession	Q56CZ1
SCOP Unique ID	n/a
Structure Solved
Class	Unknown
Molecularity	Unknown

Construct
Full Length	y
Domain	n/a
Chimera	n/a
Variants	n/a
Chain Length	352
Molecular Weight	39400.5
Pi	5.05
Molecular Weight	39400.5
Disulphides	Unknown
Full Sequence	RKDRIIMSNLFHSSSEEVGQTIPLNYTQLQYFGNITIGTPPQTFRVIFDTASNLTWVPSVGCSVEQCQDRPLYDNRHSSTYGAYGIWVTAPYIGGGAITGTAAYETITIAGINVTNQLFVEATVIDPKIYNGVQFDGSIGLSIESLYLSQHWSIFDNMFINHLLPEPVFAFYFHPTVNGSDGELILGGIEKSHYEGELTYTQSATDEWLIRLQGVKVGMRWLQTEYIYAKPVSTLPYIYGPQSQIDSINKALNASKTSGDEYTVDCSNITALPIISIKISGKNFDLRPQDYIVEVNSXCYSGFVVDEDHQQPGASTWLLGQNFLRRVYTIFKTGIFLWDKQLAFAYTRPIVY
Notes	n/a

Expression
Report	Leal AT, Pohl PC, Ferreira CA, Nascimento-Silva MC, Sorgine MH, Logullo C, Oliveira PL, Farias SE, da Silva Vaz I Jr, Masuda A. (2005) Protein Expression and Purification, 45, 107-114
Project Aim	Purification & characterization
Fusion	N-terminal hexahistidine tag
Protein Expression and Production	Protein recombinantly expressed as and refolded from inclusion bodies.
Expression Host	Escherichia coli
Expression Strain	AD494(DES)LysS
Expression Temp	37.0
Expression Time	17 h
Expression Vector	pET19a
Expression Protocol	The recombinant plasmids pET-32b/BYC and pET-19b/BYC were transformed into E. coli strain AD494 DES LysS (Novagen) and plated onto LB agar plates containing ampicillin. Single colonies were inoculated into 2.5 ml SOB medium [21] containing ampicillin (100 μg ml−1) and grown overnight at 37 °C with shaking at 180 rpm. The overnight cultures were harvested by centrifugation, resuspended in fresh medium, and used to inoculate 500 ml of SOB medium in 2-L flasks. These flasks were incubated at 37 °C with shaking at 180 rpm until an OD600 of 0.5 was reached. For induction, isopropylthio-β-d-galactoside (IPTG, Invitrogen) was added to a final concentration of 0.4 mM and the culture was grown at 30 °C for 17 h. The cell culture was centrifuged at 10,000g for 10 min at 4 °C and the cell pellet was resuspended in phosphate buffer (10 mM sodium phosphate, 150 mM NaCl, pH 7.4). For cell lysis, the suspension was frozen, thawed out and sonicated on ice.
Method of Induction	IPTG
Cell Density at Induction	OD 0.5 = 600
Cell Disruption Method	Freeze/Thaw+Sonication
Lytic Agent	None
Pre-Refolding Purification	Ion-exchange chromatography
Solubility	partial

Refolding
Refolding Method	Dialysis
Wash Buffer	50 mM Tris–HCl, 100 mM NaCl, 1 M urea, and 1% Triton X-100, pH 8.0
Solubilization Buffer	0.3% N-lauroyl sarcosine, 50 mM CAPS buffer, and 0.3 M NaCl, pH 11.0
Refolding Buffer	20 mM Tris–HCl, 150 mM NaCl buffer with gradual pH reduction (pH 10, pH 9, and pH 8)
Pre-Refolding Purification	Ion-exchange chromatography
Tag Cleaved	yes
Refolding pH	9.0
Refolding Temperature	4.0
Protein Concentration	n/a
Refolding Time	12 h
Redox Agent	None
Redox Agent Concentration	n/a
Refolding Protocol	The proteins were eluted with a serial concentration increase of imidazole (50, 100, 200, and 500 mM) in elution buffer A (0.3% N-lauroyl sarcosine, 50 mM CAPS buffer, and 150 mM NaCl, pH 11.0). Eluted fractions were analyzed by 12% SDS–PAGE. The denatured purified proteins in the eluted fractions were dialyzed in 20 mM Tris–HCl, 150 mM NaCl buffer with gradual pH reduction (pH 10, pH 9, and pH 8). Each dialysis step was performed at 4 °C during 12 h against at least 20× sample volume. Samples at pH 9.0 were collected for SDS–PAGE analysis.
Refolding Assay	Western Blot
Refolding Chaperones	None
Refolding Additives	None
Additives Concentration	n/a
Refolding Yield	n/a
Purity	n/a
Notes	n/a

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