Biomedical Engineering Reference
In-Depth Information
50.
Protein Expr Purif
. 2006
Dec;50(2):185-95. Epub 2006 Jul
4. A semi-automated large-scale
process for the production of
recombinant tagged proteins in
the Baculovirus expression
system. Schlaeppi JM, Henke M,
Mahnke M, Hartmann S,
Schmitz R, Pouliquen Y, Kerins
B, Weber E, Kolbinger F, Kocher
HP. Discovery Technologies,
Biomolecules Production Unit,
Novartis Institutes for
BioMedical Research, Bdg.
WSJ-508.2.21, CH-4002 Basel,
Switzerland.
jean-marc.
The efficient preparation of recombinant proteins at
the lab-scale level is essential for drug discovery, in
particular for structural biology, protein interaction
studies, and drug screening. The Baculovirus
insect-cell expression system is one of the most
widely applied and highly successful systems for
production of recombinant functional proteins.
However, the use of eukaryotic cells as host
organisms and the multistep protocol required for
the generation of sufficient virus and protein has
limited its adaptation to industrialized high-
throughput operation. We have developed an
integrated large-scale process for continuous and
partially automated protein production in the
Baculovirus system. The instrumental platform
includes parallel insect-cell fermentation in 10L
BioWave reactors, cell harvesting and lysis by
tangential flow filtration (TFF) using two custom-
made filtration units, and automated purification by
multidimensional chromatography. The use of
disposable materials (bags, filters, and tubing),
automated cleaning cycles, and column regeneration
prevent any cross-contamination between runs. The
preparation of the clear cell lysate by sequential TFF
takes less than 2 h and represents considerable time
saving compared to standard cell harvesting and
lysis by sonication and ultracentrifugation. The
process has been validated with 41 His-tagged
proteins with molecular weights ranging from 20 to
160 kDa. These proteins represented several families,
and included 23 members of the deubiquitinating
enzyme (DUB) family. Each downstream unit can
process four proteins in less than 24 h with final
yields between 1 and 100 mg, and purities between
50% and 95%.
