Refold - Trafficking protein particle complex subunit 6A

Refolding Record:

Protein See all refolding records for this protein...
Protein Name	Trafficking protein particle complex subunit 6A
Abbreviated Name	TRAPPC6A
SCOP Family	Unknown
Structure Notes
Organism	Human
UniProt Accession	O75865
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	158
Molecular Weight	17505.4
Pi	5.14
Molecular Weight	17505.4
Disulphides	Unknown
Full Sequence	MADTVLFEFLHTEMVAELWAHDPDPGPGGQKMSLSVLEGMGFRVGQALGERLPRETLAFREELDVLKFLCKDLWVAVFQKQMDSLRTNHQGTYVLQDNSFPLLLPMASGLQYLEEAPKFLAFTCGLLRGALYTLGIESVVTASVAALPVCKFQVIPKS
Notes	n/a

Expression
Report	Kümmel D, Müller JJ, Roske Y, Misselwitz R, Büssow K, Heinemann U. (2005) EMBO Rep., 6, 787-93
Project Aim	Structural Studies
Fusion	N-terminal hexahistidine tag
Protein Expression and Production	Protein recombinantly expressed as and refolded from inclusion bodies.
Expression Host	Escherichia coli
Expression Strain	None
Expression Temp	0.0
Expression Time	00
Expression Vector	pQTEV
Expression Protocol	The human TPC6 and TPC5 cDNAs were cloned into the bacterial expression vector pQTEV (Scheich et al, 2004) and expressed in superior broth (SB) medium with 1 mM isopropyl-1-thio-β-D-galactopyranoside.
Method of Induction	IPTG
Cell Density at Induction	OD n/a = n/a
Cell Disruption Method	Not stated
Lytic Agent	None
Pre-Refolding Purification	Ni-NTA agrose chromatography
Solubility	insoluble

Refolding
Refolding Method	Column refolding: Size-exclusion chromatography
Wash Buffer	n/a
Solubilization Buffer	8 M urea
Refolding Buffer	90 min gradient from 6 to 1 M urea
Pre-Refolding Purification	Ni-NTA agrose chromatography
Tag Cleaved	yes
Refolding pH	0.0
Refolding Temperature	0.0
Protein Concentration	n/a
Refolding Time	n/a
Redox Agent	None
Redox Agent Concentration	n/a
Refolding Protocol	TPC6 was purified with Ni affinity chromatography, after tag cleavage by the tobacco etch virus (TEV) protease (encoding plasmid was kindly provided by Gunter Stier, EMBL Heidelberg). TPC6 was subjected to size-exclusion chromatography and concentrated to 9.7 mg ml−1. TPC5 was purified from inclusion bodies under denaturing conditions (8 M urea) and refolded on a column with a 90 min gradient from 6 to 1 M urea. After dialysis against phosphate-buffered saline (PBS), folding of the protein was confirmed with CD spectroscopy. Selenomethionine-labelled TPC6 was produced according to the protocol of Budisa et al (1995). BET3 was expressed and purified essentially, as described previously (Turnbull et al, 2005). Detailed cloning and purification procedures are available as the supplementary information online.
Refolding Assay	Circular Dichroism (uv-CD)
Refolding Chaperones	None
Refolding Additives	None
Additives Concentration	n/a
Refolding Yield	n/a
Purity	n/a
Notes	The refolding temperature, pH are not stated