Plasmid Vectors for Proteomic Analyses in Giardia: Purification of Virulence Factors and Analysis of the Proteasome

• DOI: 10.1128/ec.00092-12
• Publication Date: 2012-05-20T12:55:52Z
• Authors: Jon Jerlström-Hul...; Britta Stadelmann; Sandra Birkestedt; Ulf Hellman; Staffan G. Svärd
• Keywords: Tandem affinity purification; FLAG-tag; Giardia; Expression cassette; Streptavidin; Protein purification

In recent years, proteomics has come of age with the development efficient tools for purification, identification, and characterization gene products predicted by genome projects. The intestinal protozoan Giardia intestinalis can be transfected, but there is only a limited set vectors available, most them are not user friendly. This work delineates construction suite cassette-based expression use in Giardia. Expression provided strong constitutive ornithine carbamoyltransferase (OCT) promoter, tagging possible both N- C-terminal configurations. Taken together, capable providing protein localization production recombinant proteins, followed purification novel affinity tag combination, streptavidin binding peptide-glutathione S-transferase (SBP-GST). option removing tags from purified proteins was inclusion PreScission protease site. efficiency feasibility producing purifying endogenous developed demonstrated active arginine deiminase (ADI) OCT stably transfected trophozoites. Moreover, we describe tagging, StrepTactin chromatography, compositional analysis mass spectrometry G. 26S proteasome employing Strep II-FLAG-tandem (SF-TAP) tag. first report Giardia, which will allow study specific parasite complexes.