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Expression systems Flashcards

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1
Q

What are the different expression systems?

A 
In vitro
Prokaryotes
Yeast
Fungal
Insect
Mammalian
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2
Q

What must be considered in a gene insert for expression?

A 
Optimal codons for tRNA
A promoter (or in vector) 
RBS
Secretory signal sequence
Fusion tag
mRNA stability
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3
Q

Outline the purification strategy?

A 
Add tags
Clone coding sequence
Identify regulatory region and induction
Collect cells and medium
DNase/physical destruction of Nucleic acids
Purify protein
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4
Q

What volume of protein can E.coli produce?

A

240mg/L

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5
Q

What are advantages of E.coli as a host?

A

Rapid culturing
Easy transformation
Accepts a range of vectors
Well characterised genome

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6
Q

What are the limitations of E.coli host cell?

A

No PTM
Only cDNA
Some proteins are insoluble/toxic/unstable

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7
Q

Where are recombinant proteins expressed?

A

Heterologous or homologous organisms

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8
Q

What are the advantages of yeast cell hosts?

A 
Cheap
Many types of vector
Large numbers of plasmids
Some PTM 
Homologous genes can be deleted
Constitutive or inducible expression
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9
Q

Do yeast cells accept all eukaryotic genes?

A

No

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10
Q

Are induction systems all switch derived?

A

No, some can be concentration sensitive

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11
Q

How can the T7 induction system be used?

A

Induce with IPTG
LacZ transcribes T7 polymerase in Ecoli genome
T7 polymerase targets promoter in plasmid to transcribe gene

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12
Q

When are cells auto induced?

A

OD= 0.6

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13
Q

Why is auto induction used?

A

To combat poor cell growth/decline without constant monitoring

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14
Q

Where are fusion tags added?

A

N or C terminal

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15
Q

What structure are fusion tags?

A

In frame, associated with cleavage sites

C terminal must remove stop codon

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16
Q

What are 3 examples of fusion tags?

A

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.

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17
Q

What are secretory signal sequences?

A

N
Basic
20-30 hydrophobes
Polar

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18
Q

What do secretory signal sequences cause?

A

Secretion from cell, formation of disulphide bonds

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19
Q

What target within a secretory signal sequence is targetted by proteases?

A

Polar residues

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20
Q

What are the higher eukaryotic systems?

A

insect and mammalian

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21
Q

Why are higher eukaryotic systems used?

A

functional studies, “quality not quanitity”

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22
Q

How do higher eukaryotic cultures grow?

A

Immortal colonies as adherent monolayers in a flask or in suspension of spinner flasks. Can be confluent

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23
Q

What levels of expression are used in mammalian cells?

A

Natural levels maintained by low copy numbers of the plasmid.

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24
Q

Is expression in a mammalian cell maintained?

A

No replication through mitosis thus transient but continuous antibiotic selection can drive integration

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25
What type of promoters are used in mammalian expression?
Viral or genomic
26
What type of switch are Tet systems?
An absolute
27
How are Tet systems encoded?
CMV promoter-TRE-cDNA-Poly(A) Transactivator TF on separate plasmid Either plasmid has resistance
28
How does Tet-on work?
Transactivator binds TRE in tetracycline presence to turn on
29
How does tet-off work?
Tetracycline binds transactivator to stop binding to TRE so no transcription
30
Which type of human cells are resistant to transfection?
Primary cells
31
What are the exceptional characteristics of lentivirus?
Can target all cells
32
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
33
How are lentiviral vectors produced?
Produced in E.coli as a plasmid (LentiX). Ѱ genes produce packaging into capsid using a helper plasmid
34
How are insect cells transfected?
By baculovirus
35
What type of virus is baculovirus?
A multi-capsid nucleopolyhedovirus
36
When is the protein of interest expressed in insect cells tranfected with baculovirus?
When the polyhedron coat is expressed to package multiple capsids
37
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
38
How much bacmid is required for tranfection?
Low levels to prevent toxic cell death
39
What type of insect cells are used for protein expression?
Sf9, Sf21
40
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
41
What is the name given to transposition production of bacmid?
Back-to-back
42
What function do BiP or honey bee melletin sequences have on transgenes in insect cells?
protein secretion
43
How much protein can an insect cell produce?
up to 25mg/L | 30-40% of total cell protein
44
Which organisms have been used to study differential gene expression?
Drosophilia EVE genes | C. elegans
45
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 ```
46
What cell properties does differential gene expression affect?
Specialisation, proliferation, interactions and movement
47
When are genes amplified?
Rarely but rRNA in xenopus frog eggs and structural shell proteins in drosophila eggs
48
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
49
How are xenopus rRNA genes amplified?
Excised into plasmid
50
What are examples of detection of proteins in fixed tissues?
B-gluronidase, B-galactosidase, luciferase | antibodies
51
What does EVE do?
Even skipped gene produces 7 stripe segmentation in body plan.
52
Which reporter is used in studies of EVE?
B-galactosidase to cleave Xgal into blue product
53
Which type of organisms cannot use B-galactosidase as a reporter?
plants
54
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
55
What length is the EVE regulatory region?
7.4kb upstream, 480bp crucial section
56
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
57
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
58
How is the reporter introduced into EVE?
As a plasmid for transposition when injected into pole nuclei at one cell stage
59
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
60
How does the gene insert into the mammalian genome?
Random sites, head to tail, varying copy number
61
How are recombinant embryonic stem cells selected in vitro?
Antibiotic resistance encoded in plasmid
62
How are plant cells transformed?
Using Ti plasmid from argobacterium tumefaciens
63
How doe Ti plasmid insert transgene?
T repeats excised into linear chromosome for injection and integration into plant genome
64
How are plant cells transformed with Ti recognised?
Incorporation of resistance gene into transgene to allow growth on antibiotic medium

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