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Expression system
1. Construction of a baculovirus-silkworm multigene
expression system and its application on producing
virus-like particles.
Yao L, Wang S, Su S, Yao N, He J, Peng L, et al. (2012). PLoS ONE 7(3): e32510.
BY
SAROJ KUNDAN BHARTI
PhD RESEARCH SCHOLAR (PHARMACY PRACTICE)
NIPER, Mohali
2. Introduction
Expression systems
• Expression systems are genetic constructs (a gene encoded by DNA) that are
designed to produce a proteins or RNA (ribonucleic acid), either inside or outside
a cell.
• Expression systems are used in research and in the commercial production of
enzymes or therapeutics.
• Some most commonly used protein expression system are:- E.coli, S. cerevisiae,
Baculovirus, mammalian cells expression systems.
• All of them certain advantage and limitation.
3. Protein expression system Advantages Disadvantages
E. coli • Rapid expression
• Less expensive
• Simple process scale-up
• Well characterized genetics
• No eukaryotic post-translational modifications.
• Some proteins are not properly folded
• Proteins are rarely secreted
S. cerevisiae • Moderately rapid expression
• Works well for secreted and intracellular proteins
• Less expensive
• Most protein folding and post-translational modifications are possible
• Characteristic N-linked glycan structures of proteins are
different when compared to the typical mammalian
proteins
• Requires use of yeast secretion signal peptides
Baculovirus/Insect cells • Moderately rapid expression
• Works well for secreted, membrane and intracellular proteins
• Most protein folding and post-translational modifications are possible
• Characteristic N-linked glycan structures of proteins are
different when compared to the typical mammalian
proteins
• Expensive
• Difficult to scale-up
Mammalian cells (transient
expression)
• Moderately rapid expression
• Works well for secreted and membrane proteins
• All protein folding and most authentic post-translational modifications
are possible
• Expensive
• Yields of intracellular proteins are low
• Difficult to scale-up
Mammalian cells
(stable expression)
• All protein folding and most authentic post-translational modifications
are possible
• Works well for secreted and membrane proteins
• Expensive
• Yields of intracellular proteins are low
• Difficult to scale-up
• Lengthy expression time
Table:- Comparing various protein expression systems and their respective advantages and disadvantages.
4. Core steps involved in producing the desired recombinant protein are similar across
the various expression systems.
5. • Baculovirus expression system is one of the most ideal systems for routine production of
recombinant eukaryotic proteins in insect cells, larvae and mammalian cells.
• It is widely-used in developing virus-like particles (VLPs) vaccine, displaying
heterologous peptides or proteins, and transducing genes into mammalian cells.
• Autographa californica multicapsid nucleopolyhedrovirus-Spodoptera frugiperda 9
(AcMNPV-Sf9) cell line system & Bombyx mori (silkworm) nucleopolyhedrovirus
(BmNPV)-silkworm larvae/pupae system are highly efficient baculovirus expression
system.
• Comparing with the AcMNPVSf9 system, the BmNPV-silkworm system provides
enhanced expression level and pretty low cost in silkworm larvae or pupae, which shows
promising industrialization future.
6. Multigene expression system
• Foreign DNA fragment as large as 50 kb can be accommodated into the 130 kb dsDNA
genome of baculovirus, which means several expression cassettes can be integrated into
recombinant baculovirus.
• Thus, a multiple genes baculovirus expression system (MultiBac) has been rationally
brought into the baculovirus vector for simultaneously expressing heterologous proteins.
• The MultiBac system provides a powerful tool for over-expressing the low abundance
protein complexes within cell for functional study.
8. • The efficiency of transferring genes from pCTdual to BmBacmid is 99.8%, and it
is sufficient to ensure the positive recombinant identification with almost 100%
success when combining the Gm sensitive testing.
• pUCDM-derivative integration into BaBcmid is relatively less efficient (about
93%) through cre-loxp site-specific recombination method.
• To fix this weakness, we introduced the IRES-egfp fragment into pUCDM to
construct a new donor vector pUCDMIG, which contains the 59-UTR IRES
sequence from Rhopalosiphum padi virus and a positive GFP marker.
9. Production of infective recombinant BmNPV expressing multiple
foreign genes in B. mori larvae
• The invasive DAP auxotrophic E. coli, which contains recombinant Multi-
BmBacmids carrying six foreign genes including egfp, dsRed, eyfp, vp2, vp6 and
vp7, was injected into silkworm larval hemocoel with 15 ml overnight cultures at
a 10 fold dilution.
• The hemocytes were found to be expressing GFP, DsRed and YFP simultaneously
when observed under a laser confocal microscope.
• It indicated that infective recombinant BmNPV has been generated and the three
fluorescence proteins were also expressed successfully in B. mori larvae
10. Production of rotavirus-VLPs in silkworm larvae
• Three fluorescence proteins, the three structural proteins (VP2, VP6 and VP7) of
rotavirus were also successfully expressed in silkworm larvae according to the
western blotting experiment.
• Furthermore, the round virus like particles were found in specimen, which means
VP2, VP6 and VP7 were co-expressed in silkworm larvae and had self assembled
into VLPs.
• The results above indicated multiple genes were able to be co-expressed in
silkworm simultaneously using our new BmMutiBac expression system.
• The total protein content in the purified VLPs from the hemolymph collected from
500 larvae was 6.35 mg (12.7 µg/larval hemolymph).
• This shows BmNPV-silkworm multigene expression system provides an economic
and efficient solution for producing antiviral vaccine derived from VLPs.
12. Conclusion
• Multiple expression cassettes can be introduced into the Bombyx mori nuclear
polyhedrosis virus (BmNPV) genome efficiently.
• It has great capacity of accommodating foreign genes and the low cost of feeding
silkworm, this high efficient BmMultiBac expression system provides a suitable
platform to produce VLPs or protein complexes.
