title: Expression, characterization and subcellular localization of the Mycobacterium tuberculosis PPE gene Rv1917c.
abstract: METHODS
  The PPE gene family of Mycobacterium tuberculosis is thought to be of immunological significance. One member, Rv1917c, is highly polymorphic in clinical isolates.
  
  
  OBJECTIVE
  To characterize Rv1917c gene polymorphism and expression, and to determine the cellular location and glycosylation status of the encoded protein.
  
  
  METHODS
  Tandem repeat regions of Rv1917c were amplified and sequenced to determine the molecular basis for the gene polymorphism. RT-PCR analysis was utilized to detect expression of Rv1917c mRNA in liquid cultures of M. tuberculosis H37Rv. The gene was cloned as a 3'-terminal green fluorescent protein (GFP) fusion, downstream of an acetamide-inducible promoter, and expressed in Mycobacterium smegmatis and Mycobacterium bovis BCG. The expression product was characterized in terms of cellular location and glycosylation status.
  
  
  RESULTS
  PCR and sequence data demonstrated that variable numbers of tandem repeats within Rv1917c contribute to gene polymorphism. RT-PCR analysis demonstrated that Rv1917c mRNA is expressed in liquid cultures of M. tuberculosis H37Rv. Expression of the recombinant protein in M. smegmatis and M. bovis BCG was visualized by fluorescence microscopy and flow cytometry. A protein of the predicted size (166 kDa) was confirmed by Western blotting. Cell fractionation studies demonstrated that the recombinant protein is hydrophobic, suggestive of cell wall-association, while flow cytometric data derived from antibody binding experiments suggested that it is surface exposed. Analysis of the glycosylation status of the expressed protein failed to demonstrate glycosylation.
  
  
  CONCLUSIONS
  Rv1917c mRNA is expressed in M. tuberculosis H37Rv, and Rv1917c gene polymorphism is associated with variable numbers of tandem repeats. The recombinant Rv1917c protein is surface exposed.
authors.affiliation: Department of Medical Biochemistry, University of Stellenbosch Medical School, Tygerberg, 7505, South Africa.
authors.forename: S L
authors.initials: SL
authors.lastname: Sampson
authors.forename: P
authors.initials: P
authors.lastname: Lukey
authors.forename: R M
authors.initials: RM
authors.lastname: Warren
authors.forename: P D
authors.initials: PD
authors.lastname: van Helden
authors.forename: M
authors.initials: M
authors.lastname: Richardson
authors.forename: M J
authors.initials: MJ
authors.lastname: Everett
mesh_terms: D003001:Cloning, Molecular / Q000379:methods; D005434:Flow Cytometry; D015870:Gene Expression; D006031:Glycosylation; D006801:Humans; D008856:Microscopy, Fluorescence; D009163:Mycobacterium bovis / Q000235:genetics; D020102:Mycobacterium smegmatis / Q000235:genetics; D009169:Mycobacterium tuberculosis / Q000235:genetics*; D011110:Polymorphism, Genetic; D020133:Reverse Transcriptase Polymerase Chain Reaction; D020080:Tandem Repeat Sequences / Q000235:genetics