title: Plasmid vectors for proteomic analyses in Giardia: purification of virulence factors and analysis of the proteasome.
abstract: In recent years, proteomics has come of age with the development of efficient tools for purification, identification, and characterization of gene products predicted by genome projects. The intestinal protozoan Giardia intestinalis can be transfected, but there is only a limited set of vectors available, and most of them are not user friendly. This work delineates the construction of a suite of cassette-based expression vectors for use in Giardia. Expression is provided by the strong constitutive ornithine carbamoyltransferase (OCT) promoter, and tagging is possible in both N- and C-terminal configurations. Taken together, the vectors are capable of providing protein localization and production of recombinant proteins, followed by efficient purification by a novel affinity tag combination, streptavidin binding peptide-glutathione S-transferase (SBP-GST). The option of removing the tags from purified proteins was provided by the inclusion of a PreScission protease site. The efficiency and feasibility of producing and purifying endogenous recombinant Giardia proteins with the developed vectors was demonstrated by the purification of active recombinant arginine deiminase (ADI) and OCT from stably transfected trophozoites. Moreover, we describe the tagging, purification by StrepTactin affinity chromatography, and compositional analysis by mass spectrometry of the G. intestinalis 26S proteasome by employing the Strep II-FLAG-tandem affinity purification (SF-TAP) tag. This is the first report of efficient production and purification of recombinant proteins in and from Giardia, which will allow the study of specific parasite proteins and protein complexes.
authors.affiliation: Department of Cell and Molecular Biology, BMC, Uppsala University, Uppsala, Sweden. jon.jerlstromhultqvist@icm.uu.se
authors.forename: Jon
authors.initials: J
authors.lastname: Jerlström-Hultqvist
authors.forename: Britta
authors.initials: B
authors.lastname: Stadelmann
authors.forename: Sandra
authors.initials: S
authors.lastname: Birkestedt
authors.forename: Ulf
authors.initials: U
authors.lastname: Hellman
authors.forename: Staffan G
authors.initials: SG
authors.lastname: Svärd
mesh_terms: D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics / Q000378:metabolism; D005872:Giardia / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D005873:Giardiasis / Q000469:parasitology*; D006801:Humans; D006867:Hydrolases / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D009954:Ornithine Carbamoyltransferase / Q000235:genetics / Q000378:metabolism; D010957:Plasmids / Q000235:genetics* / Q000378:metabolism; D046988:Proteasome Endopeptidase Complex / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D040901:Proteomics; D015800:Protozoan Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D037521:Virulence Factors / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism
chemical_list: D015800:Protozoan Proteins; D011993:Recombinant Fusion Proteins; D037521:Virulence Factors; D009954:Ornithine Carbamoyltransferase; D006867:Hydrolases; D046988:Proteasome Endopeptidase Complex; C019723:arginine deiminase