title: Fusion to green fluorescent protein improves expression levels of Theileria parva sporozoite surface antigen p67 in insect cells.
abstract: East Coast fever (ECF) is a fatal disease of cattle caused by the protozoan parasite Theileria parva. The development of a subunit vaccine, based on the sporozoite-specific surface antigen p67, has been hampered by difficulties in achieving high-level expression of recombinant p67 in a near-authentic form. Therefore two sets of recombinant baculovirus vectors were constructed. The first set, encoding various regions of p67, produced low levels of the corresponding p67 domains in High Five cells, despite the presence of large amounts of p67 RNA. The second, consisting of p67 domains fused to the carboxy-terminus of GFP expressed significantly higher levels of p67 protein. The GFP:p67 fusion proteins were recognized by a sporozoite-neutralizing monoclonal antibody (TpM12) raised against native p67 whereas non-fused full length p67 expressed in insect cells was not recognized. GFP-tagging therefore, appeared to enhance the stability of p67 and to conserve its folding. The high-level expression of p67 domains in a more authentic form is an important step towards the development of an effective subunit vaccine against ECF.
authors.affiliation: Laboratory of Virology, Wageningen University, Binnenhaven 11, 6709 PD, Wageningen, The Netherlands.
authors.forename: S A
authors.initials: SA
authors.lastname: Kaba
authors.forename: V
authors.initials: V
authors.lastname: Nene
authors.forename: A J
authors.initials: AJ
authors.lastname: Musoke
authors.forename: J M
authors.initials: JM
authors.lastname: Vlak
authors.forename: M M
authors.initials: MM
authors.lastname: van Oers
mesh_terms: D000818:Animals; D002460:Cell Line; D015870:Gene Expression; D017125:Genes, Protozoan / Q000235:genetics*; D005818:Genetic Engineering; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000235:genetics* / Q000378:metabolism; D011487:Protein Conformation; D015800:Protozoan Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics* / Q000378:metabolism; D018411:Spodoptera / Q000166:cytology / Q000821:virology; D034101:Sporozoites / Q000235:genetics* / Q000378:metabolism; D016797:Theileria parva / Q000235:genetics* / Q000378:metabolism
chemical_list: D008164:Luminescent Proteins; D015800:Protozoan Proteins; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; C114309:p67 antigen, Theileria; D049452:Green Fluorescent Proteins