Production of a recombinant antibody fragment in whole insect larvae.
Infection of insect cells with baculovirus expression constructs is commonly used to produce recombinant proteins that require post-translational modifications for their activity, such as mammalian proteins. However, technical restraints limit the capacity of insect cell-based culture systems to be scaled up to produce the large amounts of recombinant protein required for human pharmaceuticals. In this study, we designed an automated insect rearing system and whole insect baculovirus expression system (PERLXpress) for the expression and purification of recombinant proteins on a large scale. As a test model, we produced a recombinant mouse anti-botulinum antibody fragment (Fab) in Trichoplusia ni larvae. A recombinant baculovirus co-expressing the Fab heavy and light chains together with N-terminal sequences from the silkworm hormone bombyxin, to direct proteins into the secretory pathway, was constructed. Fifth instar larvae were reared and infected orally with recombinant (pre- occluded) baculovirus using the automated system and harvested approximately after 4 days. The total yield of recombinant Fab was 1.1 g/kg of larvae, resulting in 127 mg of pure Fab in one production run. The Fab was purified to homogeneity using immobilized metal affinity chromatography, gel filtration, and anion exchange chromatography. The identity of the purified protein was verified by Western blots and size-exclusion chromatography. Purified recombinant Fab was used to detect botulinum toxin in ELISA experiments, demonstrating that the heavy and light chains were properly assembled and folded into functional heterodimers. We believe that this is the first demonstration of the expression of a recombinant antibody in whole insect larvae. Our results demonstrate that a baculovirus-whole larvae expression system can be used to express functionally active recombinant Fab fragments. As the PERLXpress system is an automated and linearly scalable technology, it represents an attractive alternative to insect cell culture for the production of large amounts of human pharmaceuticals.
O'Connell
Kevin P
KP
U.S. Army Edgewood Chemical Biological Center, AMSRD-ECB-RT-BM, 5183 Blackhawk Road,Aberdeen Proving Ground, MD, 21010, USA.
Kovaleva
Elena
E
Campbell
James H
JH
Anderson
Patricia E
PE
Brown
Susan G
SG
Davis
David C
DC
Valdes
James J
JJ
Welch
Richard W
RW
Bentley
William E
WE
van Beek
Nikolai A
NA
D000818:Animals; D000906:Antibodies / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D016367:Baculoviridae; D001905:Botulinum Toxins / Q000276:immunology; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D007140:Immunoglobulin Fab Fragments / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D007313:Insecta / Q000254:growth & development* / Q000378:metabolism* / Q000821:virology; D007814:Larva / Q000378:metabolism / Q000821:virology; D051379:Mice; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics