naa a22 4500 528782282 20180924065513.0 cr unu---uuuuu 180924e20141111xx s 000 0 eng 10.3929/ethz-b-000093676 doi 10.1371/journal.pone.0112643 doi (ETHRESEARCH)oai:www.research-collection.ethz.ch:20.500.11850/93676 The pCri System: A Vector Collection for Recombinant Protein Expression and Purification [Elektronische Daten] [Theodoros Goulas, Anna Cuppari, Raquel Garcia-Castellanos, Scott Snipas, Rudi Glockshuber, Joan L. Arolas, F. Xavier Gomis-Rueth] PLoS ONE Open access ethresearch A major bottleneck in structural, biochemical and biophysical studies of proteins is the need for large amounts of pure homogenous material, which is generally obtained by recombinant overexpression. Here we introduce a vector collection, the pCri System, for cytoplasmic and periplasmic/extracellular expression of heterologous proteins that allows the simultaneous assessment of prokaryotic and eukaryotic host cells (Escherichia coli, Bacillus subtilis, and Pichia pastoris). By using a single polymerase chain reaction product, genes of interest can be directionally cloned in all vectors within four different rare restriction sites at the 5′end and multiple cloning sites at the 3′end. In this way, a number of different fusion tags but also signal peptides can be incorporated at the N- and C-terminus of proteins, facilitating their expression, solubility and subsequent detection and purification. Fusion tags can be efficiently removed by treatment with site-specific peptidases, such as tobacco etch virus proteinase, thrombin, or sentrin specific peptidase 1, which leave only a few extra residues at the N-terminus of the protein. The combination of different expression systems in concert with the cloning approach in vectors that can fuse various tags makes the pCri System a valuable tool for high throughput studies. CC0 1.0 Universal http://creativecommons.org/publicdomain/zero/1.0 ethresearch Goulas Theodoros joint author Cuppari Anna joint author Garcia-Castellanos Raquel joint author Snipas Scott joint author Glockshuber Rudi joint author Arolas Joan L. joint author Gomis-Rueth F. Xavier joint author PLoS ONE S.l. : Public Library of Science 9 (11), p. e112643 1932-6203 http://hdl.handle.net/20.500.11850/93676 text/html WWW-Backlink auf das Repository (Open access) 1 Journal Article ethresearch ETHRESEARCH 856 40 http://hdl.handle.net/20.500.11850/93676 text/html WWW-Backlink auf das Repository (Open access) ETHRESEARCH 700 1- Goulas Theodoros joint author ETHRESEARCH 700 1- Cuppari Anna joint author ETHRESEARCH 700 1- Garcia-Castellanos Raquel joint author ETHRESEARCH 700 1- Snipas Scott joint author ETHRESEARCH 700 1- Glockshuber Rudi joint author ETHRESEARCH 700 1- Arolas Joan L. joint author ETHRESEARCH 700 1- Gomis-Rueth F. Xavier joint author ETHRESEARCH 773 0- PLoS ONE S.l. : Public Library of Science 9 (11), p. e112643 1932-6203 BK010053 XK010053 XK010000 ETHRESEARCH ETHRESEARCH ETHRESEARCH Journal Article Open access