title: pC6-2/caspase-6 system to purify glutathione-S-transferase-free recombinant fusion proteins expressed in Escherichia coli.
abstract: Glutathione-S-transferase (GST) fusion protein expression vectors are often employed for the expression and purification of proteins in Escherichia coli. GST is then removed by site-specific proteolysis using thrombin. However, the presence of internal thrombin cleavage sites in expressed proteins can severely affect the purification of intact proteins. Cysteine-dependent aspartate-specific proteases (caspases) are efficient enzymes with defined substrate specificity. Unlike most of the proteases used for the removal of affinity tags, caspases do not leave any amino acids at the amino-terminus of cleaved proteins. We have engineered the caspase-6 site VEMD in a pGEX vector to give the pC6-2 vector. The caspase-6 can be easily removed after cleavage. Here, we describe the detailed protocol for purifying proteins using our pC6-2/caspase-6 expression and purification system. The cleavage by caspase-6 occurs in <30 min and the entire procedure can be completed in 2 d.
authors.affiliation: National Centre for Cell Science, NCCS Complex, Ganeshkhind, Pune 411007, India.
authors.forename: Prabhat Kumar
authors.initials: PK
authors.lastname: Purbey
authors.forename: P Cyril
authors.initials: PC
authors.lastname: Jayakumar
authors.forename: Milind S
authors.initials: MS
authors.lastname: Patole
authors.forename: Sanjeev
authors.initials: S
authors.lastname: Galande
mesh_terms: D053178:Caspase 6 / Q000378:metabolism*; D004926:Escherichia coli; D005822:Genetic Vectors; D005982:Glutathione Transferase / Q000378:metabolism*; D010957:Plasmids; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification*
chemical_list: D011993:Recombinant Fusion Proteins; D005982:Glutathione Transferase; D053178:Caspase 6