ABSTRACT
Using a hybrid baculovirus system, we compared the expression of 45 recombinant proteins from six categories using two models: silkworm (larvae and pupae) and an Sf9 cell line. A total of 45 proteins were successfully expressed; preparation of hybrid baculovirus was unsuccessful for one protein, and two proteins were not expressed. A similar pattern of expression was seen in both silkworm and Sf9 cells, with double and multiple bands found in immunoblotting of the precipitate of both hosts. Degraded proteins were seen only in the silkworm system (particularly in the larvae). Production was more efficient in silkworms; a single silkworm produced about 70 times more protein than 10(6) Sf9 cells in 2 ml of culture medium.
Subject(s)
Baculoviridae/genetics , Bombyx/virology , Larva/virology , Pupa/virology , Recombinant Proteins/biosynthesis , Spodoptera/virology , Animals , Baculoviridae/chemistry , Biotechnology/methods , Bombyx/genetics , Bombyx/metabolism , Cell Line , Chimerism , Electrophoresis, Polyacrylamide Gel , Female , Genetic Engineering/methods , Humans , Immunoblotting , Larva/genetics , Larva/metabolism , Peptide Fragments/biosynthesis , Peptide Fragments/genetics , Pupa/genetics , Pupa/metabolism , Recombinant Proteins/genetics , Spodoptera/cytology , Spodoptera/geneticsABSTRACT
Recombinant bovine granulocyte-macrophage colony-stimulating factor (rboGM-CSF) was produced by the baculovirus-silkworm expression system. It was purified to 98% by (NH(4))(2)SO(4), followed by a three-step column chromatography with silica gel, ion exchange resin and a metal chelate column. The specific activity of purified rboGM-CSF was 1.6-6.3 x 10(6) ED(50) mg(-1). By this method, the specific activity was raised 160-fold and 21% of the expressed rboGM-CSF was recovered.
Subject(s)
Baculoviridae/genetics , Bombyx/metabolism , Chromatography, High Pressure Liquid/methods , Chromatography, Ion Exchange/methods , Granulocyte-Macrophage Colony-Stimulating Factor/chemistry , Granulocyte-Macrophage Colony-Stimulating Factor/metabolism , Protein Engineering/methods , Animals , Bombyx/genetics , Cattle/genetics , Cloning, Molecular , Genetic Vectors/genetics , Granulocyte-Macrophage Colony-Stimulating Factor/isolation & purification , Recombinant Proteins/chemistry , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolismABSTRACT
We developed a procedure for the large-scale purification of bovine interferon-tau (boIFN-tau) by means of a silkworm-baculovirus gene expression system. Recombinant boIFN-tau (rboIFN-tau) was efficiently produced in the silkworm infected with boIFN-tau cDNA recombinant baculovirus and accumulated in the haemolymph. To establish a purification method suitable for mass production, we tried three crude purification methods, namely, an acidification and neutralization treatment (ANT), silica gel column chromatography (SGCC), and Blue sepharose column chromatography (BSCC) with a combination of Q-sepharose (QSC) and chelating sepharose column chromatographies (CSCC). As a result, the acidification and neutralization treatment was found to be the most efficient and cost effective. With this combination, we obtained 91% pure products. To confirm the applicability of the procedure for mass production, we inoculated 100 silkworms with the recombinant virus, and recovered about 4.55 mg (1.26 x 10(8)U/mg) of 91% pure rboIFN-tau by means of a combination of the ANT, followed by QSC and CSCC.