One-Step Affinity Isolation of Recombinant Protein Using the Baculovirus/Insect Cell Expression System

β-Subunit of Human Chorionic Gonadotropin Hormone and Firefly Luciferase Simultaneously Synthesized in Insect Cells Using a Recombinant Baculovirus Are Differentially Expressed and Transported

“…Although improved strategies have been developed (Kitts and Possee, 1993), one problem with using BEVS lies in the detection and identification of recombinant viruses, as only a small fraction (0.1-1%) of the viral progenies carry a replacement of the viral polyhedrin gene with the foreign gene. Alternatives to the plaque purification strategy have been proposed that include polymerase chain reaction (PCR) (Sisk et al, 1992), fluorescence-activated cell sorting (Peng et al, 1993), immunological detection (Capone, 1989;Chen et al, 1991), use of bacterial /3-galactosidase as reporters (Pennock et al, 1984;Richardson et al, 1992), etc. The problem with the immunological detection method is the limitation of antibodies available for a given foreign gene and the necessity for the entry of immunoglobulin.…”

Insect Cell Culture: Fundamental and Applied Aspects

“…Less direct methods have also been employed to identify the recombinant viruses, including: screening by nucleic acid hybridization Fung et al, 1988;Pen et al, 1989), screening for protein expression using an antibody (Manns & Grosse, 1991;Grosse & Manns, 1995), screening for enzymatic or other activity of the expressed gene, or fluorescence-activated cell sorting (Peng et al, 1993).…”

“…The cells are sonicated continuously or with a number of pulses of sonic energy of a few Watts (Stauffer et al, 1991;Peng et al, 1993) after other homogenisation or freeze thaw procedures. Baylis et al (1991) used hypotonic buffer followed by gentle sonication at 0 °C using a water bath sonicator to release EBV alkaline Dnase from Sf9 cells.…”

“…The same has been applied to insect cell cultures. Researchers have employed the process of surfactant solubilisation as the primary step in purification and isolation of some membrane associated proteins (Domingo & Trowbridge, 1988;Peng et al, 1993). Proteins have been extracted from the membrane lipid bilayer of baculovirus infected insect cells by insertion of their hydrophobic regions into detergent micelles.…”

“…It is quite often used very early in the purification process. Epitopes used for the affinity capture may be from the protein itself (Greenfield et al, 1988;Pochon et al, 1992;Singer et al, 1994), from a small tag (Furlong et al, 1988;Clark et al, 1992;Herren & Pech, 1992;Haubruck et al, 1993;Wang et al, 1994) or a fusion partner (Cooke et al, 1994;Peng et al, 1993). This allows the use of the same generic method for the recovery of many different proteins.…”