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.

Fig 1

Schematic view of the TAP tags in the constructed vectors. (A) N-terminal SBP-GST-PreScission protease cleavage site TAP tag (N-SBP-GST). (B) C-terminal PreScission Protease Cleavage site-GST-SBP TAP tag (C-SBP-GST). SBP and PreScission protease cleavage site amino acid sequences are displayed, along with the position where the tag is removed by cleavage by PreScission protease. (C) N-terminal SF-TAP tag (N-SF-TAP) with FLAG and double Strep II epitopes in boldface, as arranged in the AN vector. (D) C-terminal SF-TAP tag (C-SF-TAP) with FLAG- and double Strep II epitopes in boldface as arranged in the AC vector. FLAG and Strep II epitopes are separated by short hydrophilic sequences to improve accessibility during purification.

Fig 2

Purification of recombinant ADI from Giardia transfectants by glutathione affinity chromatography and PreScission protease cleavage. Lysis and purification were carried out at 4°C, where possible, to preserve the activity of recombinant ADI. Lane 1, Precision PLUS protein ladder; lane 2, ADI-2 transfectant lysate after sonication; lane 3, lysate cleared by centrifugation; lane 4, insoluble cell material after centrifugation; lane 5, glutathione beads after overnight incubation with cleared lysate and washings; lane 6, glutathione bead flowthrough after overnight incubation with cleared lysate; lane 7, washed glutathione beads after overnight incubation with PreScission protease and elution; lane 8, eluate recovered after overnight cleavage with PreScission protease. Samples were resolved by 10% Tris-glycine SDS-PAGE and stained with Coomassie brilliant blue.

Fig 3

Characterization of AC-Rpn11 and AN-Rpt1 transfectants. (A) Western blot of whole-cell lysates of wild-type (WT) (WB-C6) trophozoites and AC-Rpn11 and AN-Rpt1 transfectants with anti-FLAG M2 monoclonal antibody. Expression of proteins corresponding to Rpn11-C-SF-TAP (42.5 kDa) and N-SF-TAP-Rpt1 (61.9 kDa) was detected.

Fig 4

Silver-stained SDS-10% PAGE gel of fractions from purification of N-SF-TAP-Rpt1 on StrepTactin resin. Elution of bound proteins from the resin was done with 2 mM desthiobiotin. Samples were reduced and alkylated before electrophoresis. Gel slices (1 to 18) were processed and analyzed by MALDI-TOF mass spectrometry. Lane 1, Precision PLUS protein ladder; lanes 2 to 5, StrepTactin bead washes 1, 3, 5, and 7; lane 6, StrepTactin beads after elution with desthiobiotin; lane 7, StrepTactin bead elution fraction 1 (20% of eluate); lane 8, StrepTactin bead elution fraction 1 (2% of eluate); lane 9, StrepTactin bead elution fraction 2 (50% of eluate).

Fig 5

Western blot of fractions before and after StrepTactin pulldown experiments in wild-type cells (WB/C6) and SF-TAP-tagged cell lines (AN-Rpt1 and AC-Rpn11). Alpha 4 subunit: lane 1, WB/C6, cleared lysate; lane 2, AN-Rpt1transfectants, cleared lysate; lane 3, AN-Rpt1transfectants, cleared lysate; lane 4, WB/C6, elution; lane 5, AN-Rpt1 transfectants, elution; lane 6, AN-Rpt1transfectants, elution; lane 8, AN-Rpt1 transfectants, whole cell. Rpt1 subunit: lane 1, WB/C6, cleared lysate; lane 2, AN-Rpt1transfectants, cleared lysate; lane 3, AN-Rpt1transfectants, cleared lysate; lane 4, WB/C6, elution; lane 5, AN-Rpt1transfectants, elution; lane 6, AN-Rpt1transfectants, elution; lane 8, AN-Rpt1 transfectants, whole cell.