Purification and cell-free translation of mRNA coding for a variant specific antigen from Trypanosoma brucei gambiense.
Messenger RNA (mRNA) coding for a variant specific surface antigen (VSA) from Trypanosoma brucei gambiense was isolated from total trypanosomal polyribosomes by indirect immunoprecipitation. An IgG fraction of antisera to purified VSA was obtained by ion exchange chromatography and Protein A-agarose affinity chromatography. These antibodies were then subjected to affinity chromatography on a VSA-agarose column to remove non-specific IgG. Polyribosomes from the same antigenic variant of T. b. gambiense were isolated from a cell lysate and those polysomes bearing nascent VSA were bound to the IgG by gentle mixing and the complexes formed were retrieved by precipitation with fixed Staphylococcus aureus cells. The VSA-specific mRNA was separated from these complexes by dissociation of the polysomes, deproteinization, and affinity chromatography on oligo(dT)-cellulose. The mRNA isolated in this way was shown to be undegraded, active in protein synthesis and homogeneous electrophoretically. The products of the cell-free translation of this mRNA were precipitable by specific IgG but not by antiserum to a heterologous VSA. The translation product was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel exclusion chromatography. The molecular weight of the mRNA was measured by electron microscopy, agarose and polyacrylamide gel electrophoresis. Enough highly purified mRNA can be isolated in this manner to be used for hybridization analysis of the VSA gene(s).
D000818:Animals; D000954:Antigens, Surface / Q000235:genetics*; D002474:Cell-Free System; D011132:Polyribosomes / Q000032:analysis; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000235:genetics / Q000302:isolation & purification*; D014346:Trypanosoma brucei brucei / Q000032:analysis / Q000235:genetics / Q000276:immunology*
D000954:Antigens, Surface; D012333:RNA, Messenger