Expression of functional ricin B chain using the baculovirus system.
The ricin B chain (RTB) was expressed using a baculovirus expression system. The RTB coding sequence downstream of the preproricin signal sequence was inserted in the baculovirus transfer vector pM34T. After cotransfection of Spodoptera frugiperda Sf9 cells with linearized baculovirus DNA, recombinant viruses were selected, cloned and amplified. Upon infection of Sf9 cells with these recombinant baculoviruses, RTB production was revealed by immunoblotting. RTB expression using this system was optimum 72 h after infection of the cells at a multiplicity of infection of 3. RTB produced was glycosylated and had an apparent molecular mass of 34 kDa. Most of the signal sequence was removed, but the resulting recombinant RTB had a 13-residue N-terminus extension. Immunofluorescence analysis showed that this protein was located in the endoplasmic reticulum/Golgi region of the cell. RTB was not present at the plasma membrane. Secretion was enhanced by the addition of lactose to the cell-culture medium up to 50 mM. Purification was achieved from both cells and media using immobilized lactose and the lectin activity of RTB. Results obtained with the purified recombinant protein (more than 2 mg/l culture) were identical to those obtained with native RTB in all assays for biological activity; binding, internalization and reassociation with the ricin A chain to produce toxic ricin. Moreover, the RTB translocation capacity was not altered by the N-terminal peptide, showing that recombinant RTB could be used to deliver antigenic peptides to the cytosol for the induction of cell-mediated immunity.
Ferrini
J B
JB
URA 1856 CNRS, Département Biologie-Santé, Université Montpellier II, France.
Martin
M
M
Taupiac
M P
MP
Beaumelle
B
B
D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae / Q000235:genetics*; D001483:Base Sequence; D015870:Gene Expression; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D012276:Ricin / Q000096:biosynthesis* / Q000235:genetics; D018411:Spodoptera / Q000235:genetics*
D011994:Recombinant Proteins; D012276:Ricin