Expression systems Flashcards
What are the different expression systems?
In vitro Prokaryotes Yeast Fungal Insect Mammalian
What must be considered in a gene insert for expression?
Optimal codons for tRNA A promoter (or in vector) RBS Secretory signal sequence Fusion tag mRNA stability
Outline the purification strategy?
Add tags Clone coding sequence Identify regulatory region and induction Collect cells and medium DNase/physical destruction of Nucleic acids Purify protein
What volume of protein can E.coli produce?
240mg/L
What are advantages of E.coli as a host?
Rapid culturing
Easy transformation
Accepts a range of vectors
Well characterised genome
What are the limitations of E.coli host cell?
No PTM
Only cDNA
Some proteins are insoluble/toxic/unstable
Where are recombinant proteins expressed?
Heterologous or homologous organisms
What are the advantages of yeast cell hosts?
Cheap Many types of vector Large numbers of plasmids Some PTM Homologous genes can be deleted Constitutive or inducible expression
Do yeast cells accept all eukaryotic genes?
No
Are induction systems all switch derived?
No, some can be concentration sensitive
How can the T7 induction system be used?
Induce with IPTG
LacZ transcribes T7 polymerase in Ecoli genome
T7 polymerase targets promoter in plasmid to transcribe gene
When are cells auto induced?
OD= 0.6
Why is auto induction used?
To combat poor cell growth/decline without constant monitoring
Where are fusion tags added?
N or C terminal
What structure are fusion tags?
In frame, associated with cleavage sites
C terminal must remove stop codon
What are 3 examples of fusion tags?
Streptag mimicks biotin to bind streptavidin. Elutes with desthiobiotin
Histag binds nickel and is eluted with imidazole
Maltose binding proteins bind amylose and is eluted with maltose.
What are secretory signal sequences?
N
Basic
20-30 hydrophobes
Polar
What do secretory signal sequences cause?
Secretion from cell, formation of disulphide bonds
What target within a secretory signal sequence is targetted by proteases?
Polar residues
What are the higher eukaryotic systems?
insect and mammalian
Why are higher eukaryotic systems used?
functional studies, “quality not quanitity”
How do higher eukaryotic cultures grow?
Immortal colonies as adherent monolayers in a flask or in suspension of spinner flasks. Can be confluent
What levels of expression are used in mammalian cells?
Natural levels maintained by low copy numbers of the plasmid.
Is expression in a mammalian cell maintained?
No replication through mitosis thus transient but continuous antibiotic selection can drive integration
What type of promoters are used in mammalian expression?
Viral or genomic
What type of switch are Tet systems?
An absolute
How are Tet systems encoded?
CMV promoter-TRE-cDNA-Poly(A)
Transactivator TF on separate plasmid
Either plasmid has resistance
How does Tet-on work?
Transactivator binds TRE in tetracycline presence to turn on
How does tet-off work?
Tetracycline binds transactivator to stop binding to TRE so no transcription
Which type of human cells are resistant to transfection?
Primary cells
What are the exceptional characteristics of lentivirus?
Can target all cells
What characteristics are required of a viral vector for human cells?
Replication deficient
CPPT and RRE genes for stability of RNA
LTR controls transcription
CMV promoter for constitutive expression
How are lentiviral vectors produced?
Produced in E.coli as a plasmid (LentiX). Ѱ genes produce packaging into capsid using a helper plasmid
How are insect cells transfected?
By baculovirus
What type of virus is baculovirus?
A multi-capsid nucleopolyhedovirus
When is the protein of interest expressed in insect cells tranfected with baculovirus?
When the polyhedron coat is expressed to package multiple capsids
How is baculovirus engineered for transgene?
Transfer vector undergoes transposition or double cross over recombination to baculovirus, producing bacmid
Screen by PCR amplification of bacmid or transfect into cells to observe gentamicin resistance
Isolate and insert into cells for expression of protein
How much bacmid is required for tranfection?
Low levels to prevent toxic cell death
What type of insect cells are used for protein expression?
Sf9, Sf21
What components does the transfer vector have?
T7 L&R transposon sites surronding promoter, gene, poly(A) tail, Gentamicin resistance.
OriC, LacZ, ampicillin resistance genes, F1 for ssDNA
What is the name given to transposition production of bacmid?
Back-to-back
What function do BiP or honey bee melletin sequences have on transgenes in insect cells?
protein secretion
How much protein can an insect cell produce?
up to 25mg/L
30-40% of total cell protein
Which organisms have been used to study differential gene expression?
Drosophilia EVE genes
C. elegans
How can differential gene expression be measured?
mRNA abundance by microarray or northern blotting Staining of proteins on 2D gels qPCR in situ probe hybridisation reporters encoded at 1 cell stage
What cell properties does differential gene expression affect?
Specialisation, proliferation, interactions and movement
When are genes amplified?
Rarely but rRNA in xenopus frog eggs and structural shell proteins in drosophila eggs
What proof is there that genes are not lost in differentiation?
Cell nuclei of carrots and frogs can be reprogrammed to become pluripotent
Cloning of mammals
How are xenopus rRNA genes amplified?
Excised into plasmid
What are examples of detection of proteins in fixed tissues?
B-gluronidase, B-galactosidase, luciferase
antibodies
What does EVE do?
Even skipped gene produces 7 stripe segmentation in body plan.
Which reporter is used in studies of EVE?
B-galactosidase to cleave Xgal into blue product
Which type of organisms cannot use B-galactosidase as a reporter?
plants
How can the regulatory region of EVE be studied?
double restriction digests
BamH1 nicks and S1 nuclease 3’ digestion
BamH1 nicks and Bal31 digests both directions
Site directed mutagenesis
What length is the EVE regulatory region?
7.4kb upstream, 480bp crucial section
What function does the 480bp segment have in stripe 2?
Binds Hunchback and 5 Biciod to activate
Binds 3 Kruppel and 3 Giant to repress
How can the 480bp segment be purified?
DNA footprint analysis uses radioactively labelled DNA and protein protects from 1bp digestion by nuclease
Gel mobility shift assay reduces migration when protein bound and supershifted when antibody is complexed
Affinity chromatography using fragment as bait
ChIP cross links protein to DNA by formaldehyde for antibody selection
How is the reporter introduced into EVE?
As a plasmid for transposition when injected into pole nuclei at one cell stage
How are genes introduced into mammalian cells?
200-300 copies injected into pronucleus or transfection of embryonic stem cells for hybridisation into embryo for implantation
How does the gene insert into the mammalian genome?
Random sites, head to tail, varying copy number
How are recombinant embryonic stem cells selected in vitro?
Antibiotic resistance encoded in plasmid
How are plant cells transformed?
Using Ti plasmid from argobacterium tumefaciens
How doe Ti plasmid insert transgene?
T repeats excised into linear chromosome for injection and integration into plant genome
How are plant cells transformed with Ti recognised?
Incorporation of resistance gene into transgene to allow growth on antibiotic medium
