Lecture 12 Flashcards
Recombinant protein expression Commercially available vectors & how they are used
- What are the protein’s characteristics?
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
- What are the intended applications?
- 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
Will I get soluble protein if I express in E. coli
?
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
Does my protein need post‐translational modifications for structure/activity?
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
Which cell have high expression level
E. coli, Yeast, Insect cells
Characteristics of E. coli
Fast, easy but no special traits
Which one got gamma-carboxylation
mammalian cells
The sequence of the characteristics chart
E. coli, yeast, insect cells, mammalian cells
What is Baculovirus?
- 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
What are the 2 distinct virions
- 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
What are the 3 phases of infection of baculovirus
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
Cycle of the baculovirus
(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
Key feature of using baculovirus as an expression vector
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
How is the polyhedrin gene replaced?
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
How is homologous recombination achieved?
Using a ‘transfer’ plasmid…..under the control of the polyhedrin promoter
Baculovirus expression systems – V1
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
Baculovirus‐Insect
Expression ‘Vector’
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”
Baculovirus expression systems – V2
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)
What is step 2 of BAC V2
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.
What is step 3 of BAC V2
Linearise WT baculovirus DNA with RE Bsu361
Used as parental viral DNA, but cannot replicate
What is step 4 of BAC V2
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
Common insect cell lines:
- Hi‐5 insect cell line (originated from Trichoplusia ni)
- Sf9 insect cell line (clonal isolates from Spodoptera fugiperda)
- Sf21 insect cell line
Step 5 of BAC V2
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
Step 6 of BAC V2
- 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
Advantages of Baculovirus expression systems
- High levels of heterologous gene expression, complete with post translational
modifications (e.g. glycosylation and phosphorylation) - Genes are not expressed continuously because infected host cells will lyse and die during each infection cycle
- The cell lines used for AcMNPV propagation grow well in suspension cultures
- Baculoviruses have a restricted host range
Considerations for designing an insect expression system:
• 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
Expression systems in yeast
• 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
Recombinant proteins from Yeast
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
Characteristics of yeast episomal plasmid from yeast shuttle vectors
- 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
Characteristics of Yeast integration plasmids (YIp)
• Lack yeast replication origin • Propagated only through integration into the yeast genome • Integration occurs by homologous recombination
Advantages of Yeast expression system
- Eukaryotic
- Easily manipulated (and easy to grow) are cost effective
- Tightly regulated promoters
- Variety of selectable markers to choose from (e.g. URA3, HIS3, TRP1, LEU2)
- Are grown in chemically defined media
- High density production of proteins (i.e. adaptable to large scale production which is useful for NMR or crystallography)
- Available strains for secreted proteins that are glycosylated
