1. Baculovirus-Insect Cell Expression System
Insect cell system
similar posttranslational modification systems between insects and mammals
Baculovirus
Infect invertebrates
Two forms
Single nucleocapsid (virus particle)
Budding off from an infected cell
Polyhedron
Clusters of nucleocapsids (virions) trapped in a protein matrix polyhedrin
Release after cell lysis

2. Life Cycle of Baculovirus

3. Two Forms of Baculovirus
virion ~ cluster of nucleocapsids
polyhedron ~ entire package of occluded
virion
~ released into the environment
after cell lysis
polyhedrin ~ protein matrix

4. Baculovirus Expression Vector System
Host insect cell
Spodoptera frugiperda (Sf)
Bombyx mori
Baculovirus vector
Autographa californica multiple nuclear polyhedrosis virus (AcMNPV)
Promoter of the polyhedrin (polyh) gene
exceptionally strong
used for the production of heterologous protein

5. Baculovirus Expression Vector System
Transfer vector
E. coli-based plasmid carrying a segment of AcMNPV DNA

6. Baculovirus Expression Vector System
Insect cells are cotransfected with AcMNPV DNA and a transfer vector carrying a cloned gene. (Figure 7.12)
 double crossover event (loss of polyhedrin gene)
 occlusion-negative plaques
 isolation of recombinant baculovirus
(For the visual identification, lacZ under the control of a baculovirus promoter can be used.)

7. Baculovirus Expression Vector System

8. Increasing the Yield of Recombinant Baculovirus
Linearization of AcMNPV genome before infection
Reduced infectivity
Recovery of infectivity by recombination with transfer vector
Up to 99% of the baculovirus plaques are recombinant.

9. E. coli-Insect Cell Baculovirus Shuttle Vector
A. Construction of bacmid (baculovirus-plasmid hybrid molecule) by double crossover
An attachment site is inserted into the lacZ’ gene without impairing the function of lacZ’.
B. Construction of recombinant bacmid
E. coli carrying a bacmid is cotransformed with donor and helper plasmids.
Integration of donor plasmid into bacmid occurs with the help of the help plasmid (only in the presence of transposition proteins).
This integration destroys the lacZ’ function.
C. Select white colonies. (Identification of recombinant clones by lacZ’ selection)

10. E. coli-Insect Cell Baculovirus Shuttle Vector

11. Mammalian Glycoprotein and Processing of Precursor Proteins in Insect Cells
For proper glycosylation
Using mammalian α-2,6-sialtransferase and β-1,4-galactotransferase for terminal sialic acid and galactose
For processing of precursor proteins
Introduction of genes for processing enzymes (proprotein convertase)

12. Mammalian Cell Expression System
For the production of heterologous proteins with a full complement of posttranslational modifications
Chinese hamster ovary (CHO) cells are commonly used.
Cell lines
For short term expression
COS: from African green monkey kidney
BHK: baby hamster kidney
HEK-293: human embryonic kidney
For long term (stable) gene expression
CHO: Chinese hamster ovary

13. Mammalian Expression Vectors
For mammalian cell system
orieuk
promoter (p) / intron (I) / multiple cloning site (MCS) / polyadenylation (pa) sequences / transcription termination (TT) sequences
An intron enhances the production of heterologous protein.
p / SMG (selectable marker gene) / pa / TT
For E. coli system
oriE
Ampr gene

14. Translation Control Elements
5’ and 3’ UTR (untranslated regions)
Important for efficient translation and mRNA stability
Kozak sequence (K)
specific sequence surrounding the start codon (AUG)
CC(A or G)CCAUGG
for efficient initiation of translation
Signal sequence (S) ~ for secretion
Protein affinity tag (T) ~ for purification
Proteolytic cleavage site (P)
Stop codon (SC)

15. Expression of Two Genes
For the production of multimeric proteins, in vivo assembly of dimeric and tetrameric proteins is quite efficient.
Two-vector expression
Cotransfection of two plasmids
One-vector expression
Two-gene expression vector
Expression of two genes by independent promoters
Bicistronic vector
Two genes are separated by an internal ribosomal entry site (IRES) from mammalian virus

16. Two-vector expression system
Problems
Loss of one of two vectors
Unbalanced copy numbers of two vectors

17. Two-Gene Expression Vector
Under the control of two independent promoters and polyadenylation signals

18. Bicistronic Vector
Under the control of a single promoter and polyadenylation signal
to ensure the equal expression of the recombinant subunits
IRES (internal ribosomal entry site)
found in mammalian virus genomes
translation of different proteins from a polycistronic mRNA molecules

19. Bicistronic Vector

20. Selectable Markers Table 7.2
Neo (neomycin phosphotransferase, bacterial gene)
The transfected cell can grow in the medium containing G-418 (Geneticin, a neomycin derivative).
G-418: inhibitor of eukaryotic protein synthesis
Neomycin: not an effective inhibitor of eukaryotic protein synthesis
G-418 is inactivated (phosphorylated) by Neo.
Selection to increase copy number of the plasmid
DHFR-MTX (dihydrofolate reductase-methotrexate) system
GS-MSX (glutamine synthetase-methionine sulfoximine) system

21. Selectable Markers
DHFR-MTX (dihydrofolate reductase-methotrexate) system
~ DHFR- cell can not grow in the presence of MTX.
Only the transfected DHFR- cell grows in the medium
containing MTX.
~ DHFR is necessary for the production of purine.
~ MTX is an inhibitor of DHFR.
~ Selection of cells containing high copies of the vector (Fig. 7.21)
As MTX↑, DHFR↑(high copies of vectors)
(Cells only with high copies of the vector survive.)
GS-MSX (glutamine synthetase-methionine sulfoximine) system
~ GS: synthesis of glutamine, which is used in protein synthesis, purine
and pyrimidine production and other essential processes
~ MSX: inhibitor of GS
~ Transfected cells with GS gene are resistant to the toxic effects
of MSX

22. Selection of cells containing high copies of the vector