A semi-automated large-scale process for the production of recombinant tagged proteins in the Baculovirus expression system.
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 multi-step 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 multi-dimensional 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 ultra-centrifugation. 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 down-stream unit can process four proteins in less than 24 h with final yields between 1 and 100 mg, and purities between 50 and 95%.
Schlaeppi
Jean-Marc
JM
Discovery Technologies, Biomolecules Production Unit, Novartis Institutes for BioMedical Research, Bdg. WSJ-508.2.21, CH-4002 Basel, Switzerland. jean-marc.schlaeppi@novartis.com
Henke
Mario
M
Mahnke
Marion
M
Hartmann
Steffen
S
Schmitz
Rita
R
Pouliquen
Yann
Y
Kerins
Brendan
B
Weber
Eric
E
Kolbinger
Frank
F
Kocher
Hans P
HP
D000818:Animals; D016367:Baculoviridae / Q000235:genetics / Q000378:metabolism*; D019149:Bioreactors; D002478:Cells, Cultured; D010450:Endopeptidases / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D005285:Fermentation; D005822:Genetic Vectors; D015202:Protein Engineering; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification*; D018411:Spodoptera / Q000166:cytology
D011993:Recombinant Fusion Proteins; D010450:Endopeptidases