Fusion to green fluorescent protein improves expression levels of Theileria parva sporozoite surface antigen p67 in insect cells

Abstract

S U M M A R YEast 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 highlevel 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 TM 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.

Activating Transcription Factor-6 (ATF6) Mediates Apoptosis with Reduction of Myeloid Cell Leukemia Sequence 1 (Mcl-1) Protein via Induction of WW Domain Binding Protein 1

“…This strategy allowed successful detection with a fluorescent microscope. Improved expression by GFP tagging the N terminus has been observed for other proteins (28). Previous studies showed that ATF6 has a relatively short half-life (ϳ40 min) in HeLa cells.…”

Diverging Affinity of Tospovirus RNA Silencing Suppressor Proteins, NSs, for Various RNA Duplex Molecules

“…Baculovirus expressing TSWV NSs or GFP has been described previously (31,36). Recombinant baculoviruses expressing GRSV NSs and TYRV NSs were constructed using Gateway technology and the Bac-to-Bac system (Invitrogen), following the manufacturers' protocol.…”

REPAT, a new family of proteins induced by bacterial toxins and baculovirus infection in Spodoptera exigua

“…The resulting fragment was cloned into pGEM-T Easy and sequenced. The BamHI-HindIII fragment was recloned in plasmid pFBD-GFP (Kaba et al, 2002) downstream of the AcMNPV polyhedrin (ph) promoter to generate the pFBD-GFP-REPAT1 vector. This plasmid also contains the gfp gene downstream of the AcMNPV p10 promoter to facilitate screening and titration in insect cells.…”