Baculovirus Expression System

Higher Eukaryotic System

The baculovirus-insect cells system is widely used to produce recombinant proteins from a wide range of organisms. As opposed to E. coli expression system, it makes use of a higher eukaryotic cellular machinery and usually yields overexpressed proteins with proper folding and disulfide bonding. Cellular localization is usually identical to that of native protein and most post-translational modifications found in higher organisms have been shown to occur usually at the same locations. The latter cellular processings include signal peptide cleavage, N- and O-glycosilation, phosphorylation, acetylation, amidation, etc. The baculovirus system has proven to be one of the most successful aproach to produce milligram amounts of recombinant structurally- identical to the native counterpart and most often biologically active proteins.

Standard Service Description

All services include multiple steps from gene to purified protein as described in the table below.

Description Resulting Material Average turnaround (*)
Gene Synthesis Stage
-Codon optimization
-Gene synthesis
-Cloning in standard pUC plasmid
-Complete sequencing
Cloned, synthesized and verified gene 1-2 weeks
Subcloning in Baculovirus Transfer Vector Stage
-Plasmid prep
-Subcloning Bac-donor vector
-Clone selection
-Complete sequencing
Plasmid vector with cloned target gene 2 weeks
Bacmid Generation Stage
-Plasmid prep
-Recombinant bacmid selection
-Recombinant bacmid DNA isolation
-DNA Verification
Bacmid DNA with cloned target gene 2 weeks
Insect Cells Transfection Stage
-Multiple Sf9 transfection experiments
-Virus titering
-SDS-PAGE / Western blot analysis
High titer recombinant baculovirus stock 3 weeks
Pilot Expression Stage
-Expression pilot and optimization experiments
-Determine yields and protein behavior
10-50 µg of semi-purified protein 1-2 weeks
Expression and Purification Stage
-Expression
-Protein extraction
-Protein purification
-SDS-PAGE, WESTERN blot
0.1 to 3 mg of purified protein
Purity >95%
2-3 weeks

(*) Average production time for average difficult project. Actual turn around time varies with individual project features.

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