Lecture 12

Question 1

1. What are the protein’s characteristics?

Answer 1

Choice of best expression system depends on the gene involved, e.g.
• Saccharomyces cerevisiae is preferred for proteins that require significant post-translational modification
• Insect or mammalian cell lines are used when splicing of mRNA is required

Question 2

2. What are the intended applications?

Answer 2

• Structure determination experiments, e.g. X‐ray crystallography or NMR: bacterial or yeast expression are advantageous as they produce large amounts of protein
• Therapeutic target – drug interactions
• Enzyme kinetics – affect of cofactors on activity
• Protein localisation analyses

Question 3

Will I get soluble protein if I express in E. coli

?

Answer 3

Many eukaryotic proteins dont fold properly in e. coli and may form inclusion bodies, so use an eukaryotic expression system: better equipped to fold proteins from an eukaryotic source

Question 4

Does my protein need post‐translational modifications for structure/activity?

Answer 4

Many proteins need modifications after translation to become active and/or adopt the
correct structure, e.g.
-removal of N-terminal methionine residue
-N- and O-glycosylation

Question 5

Which cell have high expression level

Answer 5

E. coli, Yeast, Insect cells

Question 6

Characteristics of E. coli

Answer 6

Fast, easy but no special traits

Question 7

Which one got gamma-carboxylation

Answer 7

mammalian cells

Question 8

The sequence of the characteristics chart

Answer 8

E. coli, yeast, insect cells, mammalian cells

Question 9

What is Baculovirus?

Answer 9

• Rod shaped dsDNA virus found in many insects (Most common: Autographa californica multiple nuclear polyhedrosis virus, AcMNPV)
• Uses Spodoptera fugiperda and Trichoplusia ni moths as host insects

Question 10

What are the 2 distinct virions

Answer 10

• Occlusion derived virus (ODV) causes primary infection of host (insect feeds on plant contaminated by occluded form of the virus)
• BV is released from the infected host cells later during secondary infection

Question 11

What are the 3 phases of infection of baculovirus

Answer 11

Early (0-6h PI) virus synthesis: virus prepares the infected cell for viral DNA replication
Late (6-24h PI) BV production (extrcellular virus) Viral structural phase: Late genes that code for viral DNA replication and viral assembly are expressed
Very Late (18/24 -72 h PI) ODV form produced

Question 12

Cycle of the baculovirus

Answer 12

(A) Occlusion bodies are ingested by insect, dissolve in midgut and ODV released, which then infect epithelial cells
(B) Virion buds out of the cell and initiates systemic infection
(C) Early in systemic infection, more BV are produced which spread infection throughout insect
(D) Late in infection occluded virions are produced.
Insect dies, releasing the occlusion bodies

Question 13

Key feature of using baculovirus as an expression vector

Answer 13

Can replace the polyhedrin gene with a gene encoding a protein of interest, in vitro

Polyhedrin protein is not required for the replication of baculoviruses in cultured insect cells

Question 14

How is the polyhedrin gene replaced?

Answer 14

Homologous recombination between the polyhedrin region of AcMNPV genome and a foreign gene encoding the protein to be expressed

Homologous recombination: when nucleotide sequences are exchanged between two similar or identical molecules of DNA

Question 15

How is homologous recombination achieved?

Answer 15

Using a ‘transfer’ plasmid…..under the control of the polyhedrin promoter

Question 16

Baculovirus expression systems – V1

Answer 16

The chimeric gene (polyhedrin promoter and foreign protein coding sequence) is in
the viral genome at the polyhedrin locus in place of the wild‐type polyhedrin gene
Transfer plasmid essentials:
• MCS – to clone in cDNA
• Polyhedrin promoter - drives transciption of gene of interest
• 5’ and 3’ viral sequence - for homologous recombination

Question 17

Baculovirus‐Insect

Expression ‘Vector’

Answer 17

Essentially defined as:
“Recombinant baculovirus whose genome contains a foreign nucleic acid sequence encoding a protein of interest under the transcriptional control of the polyhedrin promoter”

Question 18

Baculovirus expression systems – V2

Answer 18

STEP 1: Clone gene of interest into MCS of transfer plasmid
FEATURES OF pBAC‐PAK9 BACULOVIRUS TRANSFER PLASMID:
Transfer plasmid landmarks:
• MCS (cloning)
• Ppolyhedrin promoter region & transcription start
• Wild‐type polh 5’UTR & 3’
sequence
Promoter: allows efficient transcription of inserted gene (drives transcription)

Question 19

What is step 2 of BAC V2

Answer 19

Purify WT baculovirus genomic DNA from budded virus progeny

use detergent disruption buffer to release cellular contents, then phenol-chloroform extraction to isolate viral DNA; followed by ethanol precipitaion of the viral DNA.

Question 20

What is step 3 of BAC V2

Answer 20

Linearise WT baculovirus DNA with RE Bsu361

Used as parental viral DNA, but cannot replicate

Question 21

What is step 4 of BAC V2

Answer 21

Mix with transfer plasmid & co‐transfect insect cells

Parental WT viral DNA undergoes homologous recombination with transfer plasmid and re-circularises as a recombinant viral DNA molecule

> able to replicate again

Question 22

Common insect cell lines:

Answer 22

• Hi‐5 insect cell line (originated from Trichoplusia ni)
• Sf9 insect cell line (clonal isolates from Spodoptera fugiperda)
• Sf21 insect cell line

Question 23

Step 5 of BAC V2

Answer 23

Identify recombinant virus by plaque assay
• Tissue culture dish contains
o Agarose
o “Grace’s” insect cell medium
o Fetal bovine serum (FBS)
layered on top of transfected cells that have adhered to the dish surface (monolayer)
• Incubate at 28°C to allow growth of cells
• Stain with neutral red stain
• Isolate an agarose ‘plug’ to grow recombinant virus in cell culture medium for over‐expression of protein

Question 24

Step 6 of BAC V2

Answer 24

• Grow host insect cells to appropriate density
• Infect with purified recombinant virus
• Incubate at 28°C for 1‐4 days
• Harvest cells by centrifugation, discard supernatant
• Lyse cells to release recombinant protein
• Purify protein

Question 25

Advantages of Baculovirus expression systems

Answer 25

1. High levels of heterologous gene expression, complete with post translational
   modifications (e.g. glycosylation and phosphorylation)
2. Genes are not expressed continuously because infected host cells will lyse and die during each infection cycle
3. The cell lines used for AcMNPV propagation grow well in suspension cultures
4. Baculoviruses have a restricted host range

Question 26

Considerations for designing an insect expression system:

Answer 26

• Select expression vector, including the style or type of promoter, that provides best
results with the recombinant gene product being expressed
• Evaluate insect cell line (e.g. Sf9, Sf21, Hi‐5 strains), growth media (serum‐
supplemented or serum‐free), and feeding/infection strategies to allow for optimal
product expression
• Choose a scalable process of cell culture and decide on other factors that may
affect/inhibit downstream processing

Question 27

Expression systems in yeast

Answer 27

• Saccharomyces cerevisiae
• Bacillus gender
• Pichia pastoris
• Methylotrophic yeast (methanol as carbon source)
• Commercial kits
• Allow stable and lasting production of proteins 
• High yield
• P. pastoris expression vectors integrate into genome
• budding yeast
• Most common yeast
• Model system
• Commonly use episomal expression 
vectors

Question 28

Recombinant proteins from Yeast

Answer 28

Protein, Use
Human Insulin, Treatment of diabetes
Hepatitis B virus surface antigen, Hepatitis vaccine
Malaria circumsporozoite protein, Malarial vaccine
Hepatitis C virus protein, Diagnostic tool
HIV‐1 antigens, Diagnostic tool
Factor XIIIa, Blood coagulation factor for therapeutic use
Fibroblast growth factor, Therapeutic: alleviate side effects of cancer treatments

Question 29

Characteristics of yeast episomal plasmid from yeast shuttle vectors

Answer 29

• Backbone of a E. coli vector such as pBR322, pUC19, pBLUESCRIPT
• Yeast selection marker(s) URA3, HIS3, TRP1, LEU2
• Yeast replication origin of the yeast 2u plasmid
• Expression of gene is controlled by GAL1 or GAL10 promoter
• High copy number: 20‐50 per cell

Question 30

Characteristics of Yeast integration plasmids (YIp)

Answer 30

• Lack yeast replication origin
• Propagated only through 
integration into the yeast 
genome
• Integration occurs by 
homologous recombination

Question 31

Advantages of Yeast expression system

Answer 31

1. Eukaryotic
2. Easily manipulated (and easy to grow)  are cost effective
3. Tightly regulated promoters
4. Variety of selectable markers to choose from (e.g. URA3, HIS3, TRP1, LEU2)
5. Are grown in chemically defined media
6. High density production of proteins (i.e. adaptable to large scale production which is useful for NMR or crystallography)
7. Available strains for secreted proteins that are glycosylated