Protein Expression

Question 1

What are the basic elements of e.coli expression plasmids?

Answer 1

• Promoter- recognised and bound by RNA pol, binding drives production of RNA transcript
• Operator- control region, binding induces conformational change of dna to allow/block access to promoter
• RBS- encodes first part of RNA transcript that is recognised and bound by ribosome
• ATG- start of gene of interest
• Gene of interest
• Stop codon indicates where translation should stop
• Terminator indicates where transcription should stop
• Replication origin
• Antibiotic resistance gene- allows selection of colonies containing plasmid of interest

Question 2

Lac operon as an example of promoter and operator function

Answer 2

• 4 proteins are encoded by 4 genes
• LacZ, lacY, lacA under control of inducible promoter
• LacI under control of consititutive promoter, lacI is always on
• LacI is a repressor, binds operator so RNA pol can’t bind to promoter
• LacZ, lacY and lacA are repressed
• Lactose acts as an inhibitor, can bind to lacI so lacI can’t bind to operator
• Can also use IPTG, a more stable analog of lactose
• Other inducible promoters can be used such as arabinose, rhamnose, galactose and tetracycline

Question 3

What is autoinduction of recombinant protein expression

Answer 3

• Don’t need to add an inducer
• Relies on consumption of a carbon source in the media
• Removes repression allowing induction
• Once carbon source is used up the cell has to switch on other metabolic pathways which can up regulate expression of the target gene

Question 4

What is Plasmid T7 promoter and regulation of T7 pol expression in recombinant protein expression

Answer 4

• Expression of recombinant proteins can be toxic
• Leaky expression – low levels of protein expression before you want it to happen
• Usually we use non-native hosts to express proteins
• Expressing high quantities of non-native protein can induce cytotoxicity if produced too early
• Can get around this by separating control of expression from cloning of gene by using non native polymerase
• Separating cloning of the gene from expression- gives more control over leaky expression
• T7 promoter is not active in E.coli lacking the T7 pol
• Not sensitive to e.coli rna pol, T7 is a phage promoter
• When ready to express you can add inducer to upregulate production of the T7 pol which has been incorporated into the chromosome
• Special e.coli strains have been generated carrying a chromosomal copy of the T7 polymerase gene on a gamma lysogen (DE3)
• When ready to express the protein decrease the temp to slow metabolism and decrease toxicity effects

Question 5

Examples of E. Coli expression strains

Answer 5

BL21- deficient in Ion and ompT proteases - if you’re expressing a heterologous protein it wont be cleaved
BL21(DE3) - carry T7 polymerase under Plac (lactose sensitive promoter), enables T7 expression, can also be deficient in Ion and OmpT proteases or deficient in RNaseE to improve stability of mRNA transcripts and increase protein expression yield
BL21(DE3)pLysS/E- same as above + plasmid pLysS or pLysE expressing T7 lysozyme to reduce basal expression of recombinant genes
BL21 trxB - deficient in Ion and ompT proteases and deficient in trxB (thioredoxin reductase), facilitates cytoplasmic disulfide bond formation

Question 6

How can protein expression be optimised

Answer 6

• Exponential phase is when cells are best used for protein production
• Usually at OD600 = 0.6 (optical density at 600nm) inducer is added so cells produce protein
• Can vary amount of protein produced by changing [inducer] and temperature
• Expression of proteins needed for bacterial life may compete with expression of target protein if resources are limited

Question 7

How do you generate gene fragments for downstream cloning

Answer 7

• Various dna polymerases can be used for PCR to generate gene fragments
• Taq pol doesn’t have proof reading activity so has high error rate
• This is not ideal
Taq can generate a single A overhang which is useful for downstream applications
Always sequence your dna to check for errors
Other examples:
Phusion high fidelity, Pfu, Deep Vent

Question 8

How to use a overhangs for cloning

Answer 8

• Incubate PCR product with Taq pol for 10 min at 75 deg C to add an A overhang
• Can use overhang to clone into plasmid
• Not directional – fragment can go in either direction

Question 9

TA cloning with topoIsomerase

Answer 9

• Topoisomerase cuts at specific sequence to form a covalent bond with 3’ phosphate
• Stable on both sides of cut site
• Ligation by removal of topoisomerase
• Not directional
• Can’t use many vectors in this way

Question 10

Traditional cloning

Answer 10

• Moving away from this
• Dependent on restriction enzymes and T4 DNA ligase
• Specific RE sites in MCS
• Do double digest with 2 different enzymes
• Design primers in PCR that allow you to clone gene of interest with extensions containing RE site
• 2 different RE sites so directional
• Ligation using T4 DNA ligase
• NOTE: extra bases added to oligomerise enable the restriction enzyme to cleave PCR product efficiently
• Facilitate binding
• Lost after digest

Question 11

Ligation-independent cloning of PCR fragments

Answer 11

• E.g. In-Fusion
• No restriction digestion, phosphatases treatment or ligation required
• PCR insert must share 15bp of identical sequence with each end of linearised vector
• Design primers to incorporate onto gene of interest regions that are identical to regions on the plasmid
• Vector is linearised
• LIC procedure using pMCSG vectors:
• Cleave vector at SspI site (AATATT, blunt end) immediately after sequence encoding tobacco etch virus (TEV) protease recognition site
• T4 polymerase in presence of dGTP removes nucleotides with 3’ to 5’ exonuclease activity until the first G, leaving a 15 base overhang
• Gene of interest (GOI) amplified by PCR using primers that begin with the complementary sequence to overhangs
• T4 pol in presence of dCTP removes nucleotides with 3’ to 5’ exonuclease activity until the first C, leaving a 15 base overhang
• Annealing between GOI and vector restores the TEV protease site in frame with the protein of interest

Question 12

Gateway technology for cloning

Answer 12

• Rapid insertion of gene of interest into vectors via DNA recombination in vitro using a recombinase

Question 13

Expression of the protein of interest as a fusion protein

Answer 13

Add tag to POI and include protease recognition site so tag can be removed after purification and POI is as close to native as possible

Question 14

What is the PolyHis tag

Answer 14

6-10 His residues
Used for purification and detection
Use a divalent metal matrix (Ni2+, Co2+)
Elite with imidazole or low pH

Question 15

Maltose-binding protein (MBP)

Answer 15

396 residues
Used for purification and enhanced solubility - keeps individual protein in solution
Binds to amylose resin
Elute with maltose

Question 16

Glutathione-S-transferase (GST)

Answer 16

211 residues
Used for purification and enhanced solubility
Glutathione agarose matrix
Elute with glutathione
GST dimerisation might affect fusion properties

Question 17

How to design forward and reverse primers to clone into expression vector

Answer 17

You can put gene of interest anywhere in the MCS
Need to make sure gene of interest doesn’t contain a restriction site
• You don’t want loads of extra foreign dna so it might be best to put the ends of the MCS
• For primers you want to end on C or g as better annealing
• For the forward primer just make the reverse complement of the 5’ to 3’ end
• For reverse primer make the reverse complement of the 3’ end and then invert it so it reads from 5’ to 3’ as all primers are synthesised from 5’

Question 18

Why are bacterial expression systems not suitable for all proteins

Answer 18

• Bacterial systems are the most popular
• Not suitable for many proteins, including eukaryotic membrane proteins
• Lack of correct folding mechanisms
• Inclusion bodies(insoluble protein aggregates)
• Incorrect post translational mods (e.g. can’t get eukaryotic glycosylation which is needed for localisation and function)
• Incorrect targeting
• Lack of appropriate lipids (e.coli don’t express any sterols)
• Loss of function

Question 19

Which expression systems are available

Answer 19

• Yeast – pichia pastoris, saccharomyces cerevisiae
• Insect – viral based system
• Mammalian – transient, stable
• Cell free
Bacterial

Question 20

Considerations when choosing an expression system

Answer 20

• Source of gene
• Specific protein requirements e.g. does it need a post trans mod
• What is the downstream application e.g. what kind of volume do you need for analysis

Question 21

How to regulate picchia pastoris expression system

Answer 21

• Pichia is methyltrophic – can metabolise methanol as it expressed alcohol oxidase (AOX)
• AOX protein is key to this ability
• AOX expression is under control of a very strong promoter
• Methanol addition upregulates AOX
• Foreign protein expression utilises AOX promoter

Question 22

Advantages of picchia pastoris expression system

Answer 22

• Pichia can grow to very high density (OD600= 80-100 vs. 1-2 in e.coli)
• Can get lots of biomass in a small volume
• Big vector

Question 23

How to use pichia pastoris expression vector

Answer 23

• E.coli vector + yeast elements (shuttle vector)
• Get gene into host vector in e.coli and amplify then transform into pichia- faster to do the earlier steps in e.coli
• All cloning done in e.coli then expression in pichia
• One ab resistance gene for e.coli and one for selection in pichia
• Not usually plasmid based expression
• Plasmid is cut prior to yeast transformation
• Integrates into host genome
• Plasmid cut in the middle of the AOX promoter
• Sticky ends
• Homologous recombination
• Can’t control how many copies of plasmid integrate into genome(multicopy incorporation)
• Need to test for optimal protein production

Question 24

How to test for high level of protein expression in pichia pastoris

Answer 24

• Growing at range of ab concs, if they grow at high conc ab they have more copies of resistance gene
• Expression level
• RtPCR tells how much dna is present in a given cell
• Monitoring of the culture is essential
• Cultured in a bioreactor
• Impeller stirs the culture according to amount of dissolved O2 in the culture, speeds up as culture grows to maintain O2 supply
• PH is monitored
• MeOH sensor, always use as little MeOH as possible as too much is toxic and flammable but you do need it to induce expression
Drop temp and pH when MeOH is added
You choose the best OD for your culture to be at when you induce

Question 25

Saccharomyces cerevisiae expression system

Answer 25

• Alternative yeast system
• Homologous recombination to generate plasmid
• Generate PCR product of gene of interest with extensions identical to regions within the chromosome
• Yeast cells have a tough outer cell wall
• Glycoprotein and chitin
• Makes cells difficult to lyse
• Constant system cell disruptor forces cell suspension through narrow nozzle under high pressure
• Causes shearing forces
• Then suspension hits a target at high speed

Question 26

Insect cell expression systems

Answer 26

• Recombinant baculovirus + cultured insect cells
• Exploit viral based expression system
• Bacmid dna contains all the virus genetic information
• Drive the production of multiple copies of baculovirus – becomes a resource
• Particles of virus coat encapsulating more bacmid DNA
• Include in the bacmid the gene of interest
• Bacmid + GOI contains all the information to produce both the complete virus particle and the protein
• Initial transfection- cells left for several; days to die and release virus particles
• Both infective and contain the bacmid + GOI
• Virus particles used to infect insect cells – incubated for 1-2 days then harvested
• At this stage the target protein should have been produced
Can determine viral titre by plaque assay
Can get controlled and reproducible protein expression

Question 27

Mammalian cell expression systems

Answer 27

• Contain all the machinery to fold and target proteins
• All shuttle vectors
• Clone in e.coli, final expression plasmid put in mammalian cell

Question 28

Cell types used for mammalian cell expression systems

Answer 28

• American type culture collection/ European collection of cell cultures
• Immortalised cell lines
• CHO cells, 3T3 cells, HeLa
• Human embryonic kidney cells give enough protein for structural analysis

Question 29

Transfection of mammalian cell expression systems

Answer 29

• Liposome mediated uptake of plasmid dna
• Mix dna with reagent that forms capsules around dna
• Need incubators with specific temp and co2 conc
• Electroporation – electric current across cells makes holes in plasma membrane
• Calcium phosphate destabilises membrane
• Only some (20% ish) of the cells will take up the plasmid

Question 30

Transient transfection of mammalian cell systems

Answer 30

• 24-48 hours post transfection cells should be expressing protein
• 49-72 hours post transfection cells containing plasmid are undergoing apoptosis
• Media starts to change colour as pH changes, cells stop adhering to the vessel
• Loss of expression
• Good for many types of experiments

Question 31

Stable transfection of mammalian cells

Answer 31

• Every cell in culture is producing POI
• Allows long term expression of POI
• Linearise dNA before transfection and it integrates into the host genome
• Selective pressure is applied through addition of an antibiotic (antibiotic resistance gene in plasmid)
• Colonies appear over time and can be checked for expression

Question 32

Cell free expression systems

Answer 32

• Mix contains components for coupled transcription and translation
• Add dna template containing sequence of POI
• Protein expressed in 1-4 hours
• Can’t really scale up
• Can tailor components
• Can include detergent micelles if it is a membrane protein
• Allows cool labelling
• You can vary components to express proteins that would not be possible in other systems

Question 33

Advantages and disadvantages of bacterial expression systems

Answer 33

Advantages:
Cheap
Quick
Flexible
Available in most labs
Easy to scale up
Disadvantages:
No PTMs
Different trafficking
Different folding systems
Non-equivalent lipids

Question 34

Advantages and disadvantages of yeast expression systems

Answer 34

Advantages:
Cheap
Quick
Available in most labs
Can perform some PTMs
Easy to scale up
Disadvantages:
Many proteins from higher eukaryotes do not express well
Non-equivalent lipids

Question 35

Advantages and disadvantages of insect cell expression systems

Answer 35

Advantages:
Quicker and cheaper than mammalian systems
Perform some PTMs
Moderately easy to scale up
Disadvantages:
Some specialised equipment required
More expensive than some other systems

Question 36

Advantages and disadvantages of mammalian cell expression systems

Answer 36

Advantages:
Ideal system for mammalian proteins
Optimised folding, trafficking and PTM systems
Disadvantages:
Some specialised equipment required
Most expensive system
Not so easy to scale up

Question 37

Advantages and disadvantages of cell free expression system

Answer 37

Advantages:
Removes the limitation of the host - can be used to easily modify a protein e.g. for NMR
Disadvantages:
Only suitable for small scale production
Expensive