The document summarizes gene cloning and expression using the baculovirus expression system in insect cells. 1. Baculovirus or Autographa californica multiple nuclear polyhedrosis virus (AcMNPV) infects insect cells like Sf9 cells and is often used as an expression vector. It uses the strong polyhedrin promoter to overexpress foreign genes. 2. The heterologous gene of interest is cloned into a transfer vector behind the polyhedrin promoter. Recombinant baculovirus is generated through homologous recombination in insect cells. 3. The baculovirus expression system allows high-level expression of properly folded and post-translationally modified

1. Gene cloning and expression in insect
cells
By : Sona Ayadi Hassan (ORCHID:0000-0003-3215-5341)
PhD student of microbial biotechnology, Faculty of Biological sciences,
Alzahra university, Vanak

2. •
• vectors and cloning strategies
• Infects insect (Sf9, e.g.) cells- Baculovirus
• Baculoviruses
• Life Cycle of Baculovirus
• Gene expression in insect cells (Baculovirus)
• Steps of Gene Expression
• Insect Cell (Baculovirus) Expression
• Protein Expression
• References

3. Baculovirus expression system
•Uses insect cells from Spodoptera frugiperda (some other species like Mamestra
brassicae and Estigmene acrea, have also been used) infected with baculoviruses
Autographa californica (multiple nuclear polyhedrosis virus AcMNPV).
•The baculovirus genome contains the gene, encoding polyhedrin, an abundant viral
protein. This protein accumulates in the insect cell towards the end of the infectious cycle
and is the major constituent of a protein matrix, containing many virions trapped
(polyhedron). Many of these polyhedrons are released into the environment after cell lysis
and the death of a single host organism.
•The promoter of the polyhedrin gene is very strong, however the gene is not essential for
the viral reproduction cycle. For these reasons it could be replaced with a heterologous
gene and this is the strategy used in the Baculovirus expression system.
•Baculovirus Expression Vector System. The transfer vector is an E.coli-based plasmid
with a segment of AcMNPV DNA.
•In vivo construction of recombinant baculovirus.
•Improvement on the original system for recombinant baculovirus production.

4. Insect Cell Expression
• Insect cells are a higher eukaryotic system than yeast
and are able to carry out more complex post-
translational modifications.
• Insect cells have the best machinery for the folding of
mammalian proteins and give the best chance of
obtaining soluble protein when expressing a protein of
mammalian origin.
• The most commonly used vector system for
recombinant protein expression in insect is
baculovirus, although baculoviral also can be used
for gene transfer and expression in mammalian cells.

5. worms infected with
recombinant baculovirus.
During infection,
the interior of each larva
liquefies.
High levels of recombinant
protein accumulate.

6. http://bti.cornell.edu/img/home/dying_caterpillar_sm.jpg
worm dying from Baculovirus

7. Baculovirus
• Baculovirus are present in invertebrates primarily insect species
• They are not infectious for vertebrates & plants
• Genome is covalently closed circular double stranded of 134
kbp, due to its small it can accommodate large fragments of
foreign DNA
• They are divided into two groups on the basis of their structure
as-:
Nucleopolyhedroviruses (NPV)
Granuloviruses
These NPV are mainly used as expression vectors i.e.
Autographa californica NPV (AcMNPV) isolated from the
larva of the alfalfa looper

8. Baculoviruses:
• DNA viruses that infect insects.
• Infection cycle involve two virus
forms:
• A: single virions: released by
infected cells and can infect other
cells
• B: polyhedron: several virions
packaged in a protein matrix
(polyhedrin)
• Polyhedrons are released after cell
death protect virions from
environmental agents

9. 1. Infects insect (Sf9, e.g.) cells- Baculovirus
• Baculovirus or Autographa californica
multiple nuclear polyhedrosis virus
(AcMNPV) many used as expression vector

10. • Baculovirus expression system based upon the ability
to propagate AcMNPV in insect cells
• Uses many of the protein modification, processing
• and transport systems present in higher eukaryotic
• cells.
• Virus that can be propagated to high titers adapted
• for growth in suspension cultures
• obtain large amounts of recombinant protein with
• relative ease
• Baculovirus are noninfectious to vertebrates and
• their promoters are inactive in mammalian cells.

11. Insects & Insect cells
• Baculovirus infects lepidopteran (butterflies & moths)
insects and insect cell lines
• Commonly used cell lines are sf9 & sf21 derived from
the pupal ovarian tissue of the fall army worm
spodoptera frugiperda and high five derived from the
ovarian cells of the cabbage looper

12. Baculovirus expression system
• Recombinant baculovirus have become widely used as
vectors to express heterologous genes in cultured
insect cells and insects larvae
• Heterologous genes placed under the transcriptional
control of the strong polyhedrin promoter of the
Autographa californica polyhedrosis virus (AcNPV)
• Based on site specific transposition of an expression
cassette (pfast Bac with gene of interest) into a
baculovirus shuttle vector (bacmid)

13. Baculovirus
Autographa californica multiple nuclear
polyhedrosis virus (AcMNPV) many used as expression vector
This is grown on insect cell lines.
Polyhedrin is synthesised in massive quantities over 4 to 5 days.
Recombinant baculoviruses overexpress foreign genes from
the polyhedrin promoter.

14. Polyhedrins may constitute up to 50% of cell protein !!!
• Polyhedron is an infective baculovirus particle (virion)
• That is protected by protein coat
• Polyhedron protects virions from sun dry ups and from
insect’s gut enzymes, it release form dead cells

15. Baculovirus Lifecycle

16. NPV infection of insect host
• Ingestion of the occlusion bodies (OB) on the diet
• the midgut of the insect
– the alkaline pH causes the dissolution of the
occlusion body releasing the virions (ODV).

17. overview of the replication cycle of baculoviruses

18. Baculovirus Protein Expression System
• Virus utilized
– AcMNPV
• Autographa californica multiple nuclear polyhedrosis virus
• A. californica = alfalfa looper
• AcMNPV infects 30 insects
• Fall armyworm - Spodoptera frugiperda (Sf9) cell line
• Polyhedrin promoter very active
– Is not essential for viral replication in BV virion
– Bombyx mori nuclear polyhedrosis virus (BmNPV)
• Silkworm and cell lines

19. Baculovirus (AcMNPV) Cloning Process
5’ 3’
Transfer vector
Polyhedrin gene
x x
Cloned gene
AcMNPV DNA
5’ 3’
Cloned gene
Recombinant
AcMNPV DNA

20. • The BmNPV bacmid system.
The expression cassette is flanked by the left and right arms of the Tn7 transposon and contains a
gentamicin resistance gene and an SV40 polyadenylation signal to form a mini Tn7

21. Steps in recombinant baculovirus
production
• Clone the gene of interest in pfast Bac donor plasmid
• Expression cassette in pfast Bac is flanked by left and right
arms of Tn7 and also an SV40 polyadenylation signal to
form a miniTn7
• Cloned pfast Bac is transformed in E.coli host strain
(DH10Bac) which contains a baculovirus shuttle vector
bacmid having a mini-attTn7 target site
• Helper plasmid which allows to transpose the gene of
interest from pfast to bacmid (shuttle vector)
• Transposition occurs between the mini-att Tn7 target site
to generate a recombinant bacmid
• This recombinant bacmid can now be used to transfect
insect cell lines.

22. The expression cassette is flanked by the left and right arms of the Tn7 transposon and contains
a gentamicin resistance gene and an SV40 polyadenylation signal to form a mini Tn7. …
Gene of Interest
Tn7R p10 Gent+ Tn7L
Gene construct
Gene of Interest
Tn7 R
PpH Tn7 L
pfast Bac with insert

24. Making proteins with baculovirus
insect cellRecombinant
protein
gene
Baculovirus
DNA
+

25. Insect Medium
• Grace’s Insect medium- unsupplemented but
contains L-glutamine
• Grace’s Insect medium supplemented-contains
additional TC yeastolate & Lactalbumin
hydrolysate
•

26. • Insect cell lines are generally considered
simple to sustain compared to mammalian cell
lines and can be maintained as either
suspension cultures, in shake or stirred flasks,
or in monolayer cultures in T-flasks or culture
dishes. Most insect cell culture medium
utilises a phosphate buffering system rather
than the carbonate-based buffers commonly
used for mammalian cell lines, negating the
requirement for CO2 incubators. The range for
growth and infection of most cultured insect
cells is between 25ºC to 30ºC, although 28ºC
is generally considered the optimal growth
temperature.

27. Requirements for proper cell culture
• Temperature- Optimal range is 27-28 C
• pH- Optimal range is 6.1 to 6.4
• Aeration-Requires passive 02 diffusion for optimal
growth & recombinant protein expression
• FBS- Working with suspension culture it is
advisable to use (10-20% FBS) to gave protection
from cellular shear forces

30. Polyhedra under light microscope
Partially dissolved polyhedron containing
bundles of virus particles.

31. Insect Cell Expression
• Insect cell expression is optimal for glycosylated protein
expression in a cost-effective manner.
• There are many advantages to using baculovirus for
heterologous gene expression. Heterologous cDNA is
expressed well.
• Proper transcriptional processing of genes with introns occurs
but is expressed less efficiently. As with other eukaryotic
expression systems, baculovirus expression of heterologous
genes permits folding, post-translational modification and
oligomerization in manners that are often identical to those that
occur in mammalian cells. The insect cytoplasmic environment
allows proper folding and S-S bond formation, unlike the
reducing environment of the E. coli cytoplasm.

32. Insect Cell Expression
• Post-translational processing identical to that of mammalian
cells has been reported for many proteins. These include proper
proteolysis, N- and O-glycosylation, acylation, amidation,
carboxymethylation, phosphorylation.
• Proteins may be secreted from cells or targeted to different
subcellular locations. Single polypeptide, dimeric and trimeric
proteins have been expressed in baculoviruses.
• Finally, expression of heterologous proteins is under the control
of the strong polyhedrin promoter, allowing levels of
expression of up to 50% of the total cell protein.

33. The perfect system E. coli Yeast Insect
-non-pathogenic
-fast growth
-inexpensive
-high expression level
-regulatable
-properly modified protein
-easy to purify product
-yes
~20 minutes
-yes
-up to gm/L
-yes
-minimal
-possibly
-yes
~2 hours
-yes
-mg/L
-yes
-some
-easy
-yes
~24 hours
-no
-mg/L
-yes
-much
-easy

34. Insect Cell Expression
The major advantages of insect cell
expression:
Eukaryotic post-translational modification
Proper protein folding and function
High expression levels
Easy scale up with high-density suspension
culture
Safe to handle

35. Transfer vector
Baculovirus
genome
Foreign gene
Polyhedrin gene
The target gene is introduced into the baculovirus genome by
homologous recombination.

36. Choice of the expression system
Cell-free Bacteria Yeast Insect Mammalian
Easy of use
Cost of media and Equipment
Pos-translational Modifications
(Probability of protein function)
Time Requirement

37. Advantages
The polyhedrin gene is not required for the continuous
production of infectious virus in insect cell culture. Its sequence is
replaced with that of the heterologous gene.
The polyhedrin gene promoter is very strong. This determines a
very high level of production of recombinant protein.
Very late expression allows for the production of very toxic
proteins.
This system is capable of post-translational modifications.

38. Characteristics of the virus

39. Structural Genes
• Three main structural genes:
– Group Specific Antigen (Gag)
– Envelope (Env) gene codes for envelope proteins
gp160, gp120 and gp41
– Polymerase (Pol)
Responsible for conversion of viral RNA into
DNA, integration of DNA into host cell DNA and
cleavage of protein precursors.

40. Life Cycle
• (a) HIV (red) attaches to two cell-
surface receptors (the CD4
antigen and CXCR4 ).
• (b) The virus and cell membrane
fuse, and the virion core enters the
cell.
• (c) The viral RNA and core
proteins are released from the
virion core and are then actively
transported to the nucleus.
• (d) The viral RNA genome is
converted into double-stranded
DNA through an enzyme unique
to viruses, reverse transcriptase
(red dot).
• (e) The double-stranded viral
DNA moves into the cell nucleus.

41. Approved HIV Drugs
• Entry Inhibitors
These drugs stop (inhibit) HIV from entering a
CD4 cell. There are different types of entry
inhibitors: fusion inhibitors and CCR5 antagonists.
One of each type is approved:
• Fusion inhibitor: Fuzeon
• CCR5 antagonist: (maraviroc)

42. pWPXL: proviruse; consist of GFP gene and site
for foreign gene.
psPAX: packaging vector; consist of gag, pol and
env genes.
pMD2G:envelope vector; consist of vsvg gene for
the envelope of psudovirus.
Human Immunodeficiency Virus pseudotype

43. pWPXL

44. psPAX

45. pMD2G
Pseudotyping with VSV-G allows transduction of a wide range of cell
types from various species, including the mouse, that HIV-1 does not
normally infect
vesicular stomatitis virus glycoprotein

47. • Murine leukemia virus "MLV"
• Murine leukemia virus Retroviridae
• Species:Murine leukemia virus
• The murine leukemia viruses (MLVs or MuLVs) are
retroviruse named for their ability to cause cancer in
murine(mouse) hosts. Some MLVs may infect others.
Replicating MLVs have a positive sense, single-stranded
RNA (ssRNA) genome that replicates through a DNA
intermediate via the process of reverse transcription.

48. Applications of MLVs
• Gene therapy: MLV-derived particles can
deliver therapeutic genes to target cells.
• As a model for Anti viral activity of
medical plant
• Reverse Transcriptase from MMLV used in
Biotechnology