title: Expression and purification of functional, recombinant Trypanosoma cruzi complement regulatory protein.
abstract: The complement regulatory protein (CRP) of Trypanosoma cruzi is a developmentally regulated glycosylphosphatidylinositol (GPI)-anchored membrane protein that protects the parasite from complement-mediated killing, and is an important virulence determinant of the microorganism. CRP binds human complement components C3b and C4b to restrict activation of the complement cascade. Here, we report production of functional, recombinant T. cruzi CRP in mammalian cells and a one-step purification of the recombinant protein. Exchange of the crp DNA sequence encoding the carboxy-terminal GPI signal sequence with the corresponding sequence of decay accelerating factor (DAF) was necessary for recognition, cleavage, and addition of GPI in mammalian cells. CRP production was assessed in two mammalian cell lines with crp-daf gene expression driven by three different transcription control regions: Rous sarcoma virus long terminal repeat, cytomegalovirus (CMV) immediate early gene, and chicken beta-actin promoter/CMV enhancer. We present evidence that CRP produced in transfected Chinese hamster Ovary (CHO) cells was functional and protected the cells from complement-mediated lysis. To facilitate purification of the recombinant protein, a hexahistidyl tag was incorporated at 3(') end of the cDNA upstream of the GPI anchor addition sequence. An additional histidine fusion construct was made that allowed for secretion and recovery of recombinant protein from culture supernatant fluid. Both membrane and secreted forms of the protein were purified in one step by nickel nitrilotriacetic acid. The production and purification of functionally active CRP in a non-infectious expression system will allow for structure and function studies aimed at identifying the active site(s) of this protein.
authors.affiliation: Department of Molecular Genetics and Biochemistry, University of Pittsburgh, Pittsburgh, PA, USA.
authors.forename: Margaret
authors.initials: M
authors.lastname: Beucher
authors.forename: Wendell S F
authors.initials: WS
authors.lastname: Meira
authors.forename: Vasthy
authors.initials: V
authors.lastname: Zegarra
authors.forename: Lúcia M C
authors.initials: LM
authors.lastname: Galvão
authors.forename: Egler
authors.initials: E
authors.lastname: Chiari
authors.forename: Karen A
authors.initials: KA
authors.lastname: Norris
mesh_terms: D000818:Animals; D016466:CHO Cells; D002522:Chlorocebus aethiops; D006224:Cricetinae; D005260:Female; D015870:Gene Expression; D006639:Histidine; D008562:Membrane Glycoproteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification*; D015800:Protozoan Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification*; D014349:Trypanosoma cruzi / Q000737:chemistry / Q000235:genetics*
chemical_list: D008562:Membrane Glycoproteins; D015800:Protozoan Proteins; D011993:Recombinant Fusion Proteins; C103951:complement regulatory protein, Trypanosoma cruzi; D006639:Histidine