Structural Biology - Protein Expression

Question 1

Explain the genetic elements involved in the production of a plasmid encoded protein

Answer 1

Promotor: DNA sequence bound by RNA polymerase. Drives production of RNA transcript
Operator (downstream of promotor): Control region that binds specific molecules. Leads to a conformational change in DNA that prevents/allows access of RNA polymerase to the promotor
Ribosome binding site: section of DNA that encodes first part of RNA transcript
ATG start site: start of gene of interest
Gene of interest
Stop codon: where translation should stop
Terminator region: where transcription should stop

Question 2

What are the extra genetic elements in the expression of a plasmid encoded protein other than the part that produces the RNA transcript?

Answer 2

Antibiotic resistance genes: selection of bacterial colonies that contain the plasmid of interest
Replication origin: allows amplification of DNA plasmid within cells

Question 3

Explain the Lac Operon

Answer 3

Expression of proteins LacI, LacZ, LacY, LacA
LacI is under control of a constitutive promotor
LacI is a repressor (RNA polymerase can’t bind) that controls expression of other Lac proteins by binding to the operator
Lactose IPTG (analogue of lactose) binds to LacI and acts as an inhibitor by inducing a conformational change so no longer binds to operator
Then get transcription and translation of 3 lac proteins
Proteins like LacI are sensitive to sugars (arabinose, galactose) or tetracycline which can be used to drive the production of a protein

Question 4

Describe the stages of cell growth (OD vs time graph)

Answer 4

Lag Phase (hour 1): inoculate culture containing low number of cells, low growth rate
Exponential phase: rapid growth, inducer molecule added at OD = 0.6
lactose added to induce expression of a target gene
Stationary phase: cells still growing but also dying rapidly, no net growth, resources used up
Death phase

Question 5

How can expression of cells be optimised?

Answer 5

Vary concentration of inducer molecule
Vary temperature of growth (18 vs 37 degrees)

Question 6

What is autoinduction?

Answer 6

Does not rely on the addition of an inducer molecule
Medium contains glucose and lactose
Glucose (or a specific carbon source) inhibits expression of target gene but as it is consumed, lactose becomes primary carbon source
Induction of target protein

Question 7

What is cytotoxicity?

Answer 7

Expression of recombinant proteins can be toxic because a small amount of protein is expressed before the culture is ready
Avoid toxicity by separating cloning of gene from gene expression

Question 8

Explain how T7 polymerase expression is regulated

Answer 8

T7 polymerase is provided by a separate plasmid (expression plasmid).
It contains the gene of T7 polymerase under control of a different promotor, like Lac
Induce Lac promotor by adding IPTG, leading to expression of polymerase
T7 polymerase binds to promotor pT7 to produce target molecule

Question 9

List examples of E. coli expression strains

Answer 9

BL21: deficient in proteases, better at producing non-native proteins
BL21/STAR: Deficient in RNase, improves stability of mRNA transcripts
BL21(DE3): Reduces expression of recombinant genes, lowers amount of target protein produced before you want it to be
BL21-AI: Different promotor, fine control of protein produced
BL21 Codon: expresses codons that are usually not expressed in E. coli
BL21 trxB: helps express a disulphide bond in cytoplasm

Question 10

Explain the properties of DNA polymerase in PCR?

Answer 10

Taq polymerase lacks 3’-5’ exonuclease activity (proofreading) = high error rate, must sequence product at end to check for errors
Taq adds a single A base overhang at 3’ end, useful for cloning
Relative processivity is high (amount of nucleotides added before polymerase dissociates
Polymerases with proofreading activity are slower

Question 11

How does A-overhang cloning work?

Answer 11

PCR product is blunt ended
Incubate PCR product with Taq polymerase at 75 degrees
Additional A base attaches at 3’ end
A overhang is used to clone PCR product into a plasmid with a T overhang due to complementarity
Non-directional insertion

Question 12

How does Topo TA-Cloning work?

Answer 12

Topoisomerase I cuts DNA at specific CCCTT sequence
Forms a covalent bond with phosphate group of last T base
Stable interaction so no reformation of the bond
Topo TA cloning vector is fixed with Taq amplified PCR product that has an additional A base
A bases attack bond between T base and topoisomerase in vector
Topoisomerase is released
Non-directional

Question 13

How does traditional cloning work?

Answer 13

Plasmid contains restriction sites in the MCS
Design primers with extensions encoding the restriction sites
ex. Nco1 site at 3’ and HindIII site at 5’
Double digest both plasmid and PCR product with restriction enzymes
Produces sticky ends so plasmid doesn’t undergo self ligation
Directional insertion

Question 14

How does ligation independent cloning work?

Answer 14

No restriction digestion
Inserts are amplified by PCR
Design primers that incorporate onto end of gene of interest. Must share 15bp of identical sequence with each end of vector
Linearise vector by PCR

Use T4 DNA polymerase in presence of dGTP (removes bases until reaches first G base to produce overhang)
Repeated with gene of interest and dCTP to create overhangs that overlap

Question 15

How does gateway cloning work?

Answer 15

Rapid insertion
Using DNA recombination in vitro and a recombinase
Can use different expression systems - can clone gene into different vectors depending on the function (ex. purification, antibody production)

Question 16

Explain the expression of a protein of interest as a fusion protein

Answer 16

Protein of interest is fused to a tag
Incorporate protease recognition site to be able to cleave off the tag

Question 17

What are some commonly used tags?

Answer 17

His tag, Maltose binding protein, Glutathione S-transferse, GFP

Question 18

Maltose binding protein

Answer 18

Purification
Enhanced solubility
Very big
Amylose resin/maltose matrix

Question 18

What are some features and their functions in an E. coli expression vector?

Answer 18

T7 promotor: T7 polymerase binds to
RBS: Ribosome binds
ATG: translation initiation
6x His Tag: detection of recombinant protein
Epitope tag: Detection of fusion protein
Enterokinase cleavage site: Site for removal of fusion tag
MCS: insertion of gene of interest

Question 19

His Tag

Answer 19

Used for detection and purification
short tag so low influence on fold of overall protein
divalent metal (Ni2+)/imidazole or low pH matrix

Question 20

GST

Answer 20

Purification
Enhanced solubility
Quite big
Glutathione agarose matrix
GST dimerisation might affect fusion properties

Question 21

GFP

Answer 21

Used for detection

Question 22

How do you design a forward primer (5’ to 3’)?

Answer 22

At start of DNA sequence
Take start of 5’ to 3’ strand
Tag on additional DNA that encodes a restriction site (BamHI) at 5’ end
End on a C or G base (3 H bonds increase annealing)

Question 23

How do you design a reverse primer?

Answer 23

At end of DNA sequence
Consider 5’ to 3’ strand
Use complement of strand to create 3’ to 5’ strand
Tag on to the 5’ end the sequence encoding restriction site for other enzyme (HindII)
Add additional sequence at 5’ on both reverse and forward primer to allow enzyme to bind and cleave (GCG)

Question 24

How do you isolate a His tag using metal affinity chromatography?

Answer 24

Bind protein of interest to Ni2+ NTA column Washing steps to remove any non specific proteins Elute with imidazole Proteolytic degradation to remove the His-tag Load material on a gel Get 2 bands (one his tag, one non his tag protein) Use non his tag protein to grow crystals

Question 25

what are the disadvantages of bacterial expression systems?

Answer 25

Not suitable for many proteins: lack correct folding mechanisms Can form inclusion bodies: insoluble aggregates resulting from incorrect folding Incorrect post translational modifications: No eukaryotic forms of glycosylation Incorrect targeting of protein in the cell Lack appropriate lipids: no cholesterol, E coli doesn't express any sterols Loss of function

Question 26

What expression systems are available?

Answer 26

Yeast: Pichia pastoris, Saccharomyces cerevisia Insect: Viral based system Mammalian: Transient, Stable Cell free

Question 27

What considerations do you have to make to choose an expression system?

Answer 27

Source of gene: Plant protein/mouse protein/human protein Specific protein requirements: Ex. If it needs a post translational modifications What is the downstream application: Volumes of material that needs to be expressed

Question 28

Explain the Pichia Pastoris expression system in yeast

Answer 28

Pichia can metabolise methanol (methylotrophic) AOX1 promotor controls AOX (alcohol oxidase) protein expression AOX1 promotor is induced by methanol leading to protein production Use of a shuttle vector allows cloning in E. coli but expression in Pichia

Question 29

What is a shuttle vector?

Answer 29

Used to move DNA sequences between different types of cells (ex. bacterial and eukaryotic) Can replicate in multiple hosts

Question 30

What is the Pichia Pastoris system used for?

Answer 30

GFP pichia expression vector is linearised SacI cuts plasmid in the middle of the AOX promotor to produce a sticky overhang Vector undergoes homologous recombination reforms correct length of AOX promotor Inserted into host genome AOX1 promotor is placed in front of the gene of interest so it is expressed when methanol inducer is added

Question 31

How is a bioreactor used to monitor pichia pastoris cell culture?

Answer 31

Impellor stirs the culture according to amount of dissolved oxygen As oxygen drops, it speeds up Monitor pH Monitor temperature Methanol sensor Need to add enough to induce expression of target protein But too much methanol is flammable and toxic DO2 initially high, DO2 decreases over time as cells grow Add methanol slowly At same time drop temperature and Increase pH

Question 32

How is Saccharomyces cerevisiae used as an alternative yeast system?

Answer 32

Generate expression plasmids using homologous recombination No restriction site cloning like with E coli Generate PCR product that incorporates extensions that are identical to regions in the vector Mix PCR product extensions with linearized vector Insertion of gene of interest

Question 33

How can protein production be measured in the cell?

Answer 33

Fluorescence Perform localisation analysis

Question 34

How do you break the yeast cells?

Answer 34

Yeast cells have a tough cell wall made of glycoprotein and chitin Makes cells difficult to lyse Need to be able to lyse cells to obtain cellular protein Constant system cell disruptor (french press) High pressure Causes shear forces that disrupt the cell wall and membrane

Question 35

How do insect cell systems work?

Answer 35

Recombinant Donor plasmid contains regions for homologous recombination, gene of interest and a specific Insect vector promotor Mix plasmid with bacmid DNA Integration of gene of interest with it's promotor into the bacmid Transform into E coli cells Antibiotic selection Extract recombinant bacmid DNA Perform initial transfection Insert DNA into insect cells Leave cells to die for 3-4 days Produce recombinant virus particles and use them for further rounds of infection of insect cells Incubated for 1-2 days Determine viral titer via plaque assay: Know how much virus stock to add for reproduction of experiment by using virus particle stock to infect cells over a shorter period of time Cells contain the bacmid + the GOI Target protein is produced

Question 36

What is bacmid DNA?

Answer 36

Baculovirus is used as a vector to introduce gene of interest into insect cells Contains all of the virus genetic material Allows production of multiple copies of baculovirus Particles of virus coat encapsulating more bacmid DNA: used for multiple rounds of infection

Question 37

Advantages of insect cell systems

Answer 37

Easy to culture cost efficient can perform post-translational modifications so suitable for the expression of complex proteins high yields of recombinant proteins

Question 38

Cell types in mammalian cell systems

Answer 38

All vectors are shuttle vectors All cloning occurs in E coli Only when final expression plasmid is produced it is moved to mammalian cells Human embryonic kidney cells Immortalised cell lines (HeLa cell line first immortalized cell line, CHO cells, 3T3 cells)

Question 39

What are the different types of transfection?

Answer 39

Transfection: how DNA is transported into the cell Liposome mediated uptake of plasmid DNA: Mix DNA with a reagent that forms capsules around DNA (lipofection reagent), DNA lipid complex is endocytosed into the cell, endosome is broken down and DNA is released into the cell. Electroporation Calcium phosphate treatment Only some cells will take up the plasmid

Question 40

What is transient transfection?

Answer 40

24 - 48 hours after transfection cells should be expressing the protein 48 - 72 hours after cells containing plasmid undergo apoptosis Loss of expression Good for many different types of experiments

Question 41

What is stable transfection?

Answer 41

Long term expression of the protein of interest Linearise DNA before transfection Integration of DNA into host genome Selective pressure is applied through addition of antibiotic - only cells that have taken up the plasmid will survive Colonies appear over time (several weeks) and can be checked for expression Initially only have small number of cells

Question 42

What is cell free expression?

Answer 42

Non native hosts used most of the time (except mammalian cell systems) which causes problems Cell free expression: Contains all elements you need to produce a protein without the cell Add PCR fragment or plasmid DNA Incubate mixture Can vary components to express proteins that would not be possible in other systems

Question 43

Bacterial cell system advantages disadvantages

Answer 43

A: Cheap, quick, flexible, available in most labs, easy to scale up D: no PTMs, different trafficking, folding systems, non-equivalent lipids

Question 44

Yeast cell system advantages disadvantages

Answer 44

A: Cheap, quick, flexible, available in most labs, easy to scale up, can perform some PTMs D: many proteins from higher eukaryotes do not express well, non-equivalent lipids

Question 45

Insect cell system advantages disadvantages

Answer 45

A: quicker and cheaper than mammalian, perform some PTMs, easy to scale up D: some specialised equipment needed, more expensie

Question 46

Mammalian cell system advantages disadvantages

Answer 46

A: ideal system for mammalian proteins, optimised folding, trafficking and PTMs systems D: some specialised equipment needed, most expensive system, not easy to scale up

Question 47

Cell free system advantages disadvantages

Answer 47

A: removed the limitation of the host - can be used to easily modify a protein eg. for NMR D: only suitable for small scale, expensive

Question 48

Purposes of protein engineering

Answer 48

Probe mechanism: High resolution structure doesn't tell us how the protein works, make a change in the molecule to probe how it works Create novel proteins: Function in extreme conditions Improve catalytic function (Kcat or Kcat/Km) Alter substrate specificity or stereospecificity: Change binding site to broaden specificity Improve stability: Membrane proteins are very unstable when isolated --> makes it hard to find structure Requirements growing as biotech applications increase: More eco friendly production processes (ex. More efficient crops = less land used), Dealing with ecological challenges (eg. Plastic and other contamination issues)

Question 49

What does protein engineering rely on?

Answer 49

Protein engineering relies on mutagenesis - either targeted changes to the protein or random mutagenesis (directed evolution). Sometimes combinations of these can be used

Question 50

Explain the synthetic gene route for mutagenesis

Answer 50

To make many single point mutations at the same time OR add tags/cleavage sites Generate electronic file of sequence of gene with all the substitutions Send it to a company which will generate the synthetic gene This would take a lot of time in the lab (must make mutations one by one)

Question 51

Explain the plasmid based PCR approach for mutagenesis

Answer 51

Gene in plasmid with target site for mutation Design mutagenic primers that contain the desired mutation and have DNA extensions Denature plasmid Mix plasmid with mutagenic primers and carry out PCR Get parental strands and daughter strands in the PCR mixture Parental strands are methylated and unmutated so are digested by DpnI Transform daughter cells into E. coli

Question 52

Explain the overlap extension methods using PCR

Answer 52

Identify target site for mutation Design mutagenic primers that contain the substitution and have extensions on both sides Two restriction sites: one upstream and one downstream of the mutation Oligonucleotide primers that are complementary to RS Carry out two separate PCR reactions PCR with Oligo 1 + 4: Produce fragment that has RS at 5' site and mutation at 3' end PCR with Oligo 2 + 3: Produce second fragment that has mutation at 5' end and second RS at 3' end Purify, mix, denature, anneal Get overlap of more than 20 bases PCR reaction 3 includes oligo 1 and 2: DNA is annealed at the overlap site Cut fragment with RE and replace wild type fragment

Question 53

What is alanine scanning mutagenesis

Answer 53

Systematic approach to engineering proteins Used to investigate role of AA residues Obtain a more stable construct Every residue is mutated to Alanine Except pre-existing Alanine that is mutated to Leucine Used to focus on a particular region in the protein Need to have a means of assessing effects of mutations like using a Functional assay But can knock out function of the protein by mutagenesis: so must analyse by other means (3D structure)

Question 54

What are random mutagenesis approaches

Answer 54

Chemical mutagenesis: treat plasmid with chemical (ex. sodium bisulfites, nitrous acid, hydrazine, dimethyl sulphate) that damages DNA Sloppy/error-prone PCR: increase levels of Mn2+, change ratio of dNTP by increasing dGTP to introduce errors, mutation rate is 0.6-2% Plasmid in E. coli cells: chemical (ethane methyl sultanate) or UV/X-rays to introduce mutations, use in repair-defective strains of E. coli (mut mutants) DNA shuffling: in vitro recombination, fragment with DNAse I and use as template in PCR

Question 55

Explain the iterative (repetitive) process of mutagenesis

Answer 55

Wild type enzyme Isolate wild type gene Mutagenesis Express mutant genes Selection of functional mutant genes (not all functional) Isolation of genes for improved enzymes Use mutant genes for subsequent rounds of mutagenesis

Question 56

How to screen for/detect variants of a protein

Answer 56

A) Gas/liquid chromatography or UV. SLOW B) Altered substrate: produce a fluorescent biproduct. But altered substrate will bind in a different way = not good idea C) Less direct methods: Fluorescent cofactor, Coupled reaction (Product used by Enzyme 2 to create fluorescent biproduct)

Question 57

Explain iterative saturation mutagenesis (ISM) for rapid directed evolution

Answer 57

Combination between targeted and random mutagenesis Focus on specific region that is causing instability Gene encoding protein is mutated to create a library of variants Make all possible amino acid substitutions at a specific site in the protein library of variants is screened for desired function best performing variants are used for next round of mutagenesis, steps are repeated several times until optimised protein is achieved

Question 58

Case study of bacillus subtilis for iterative saturation mutagenesis (ISM)

Answer 58

Making lipase of Bacillus subtilis more thermostable Specific regions identified as contributing to low stability due to high B-factors Specifically targeted these regions to increase the melting temperature (when 50% of protein is unfolded, the higher, the better) Some variants had increased melting temperature Chose best one and produced more variants with significantly increased melting temperature

Question 59

How to test for mutants after mutagenesis

Answer 59

Functional analysis Thermostability analysis Antibiotic resistance

Question 60

1) What is UapA?

Answer 60

Uric-acid xanthine transporter from Aspergillus nidulans High affinity, high capacity H+ symporter - couples movement of substrate and protons along concentration gradient Potential antifungal drug target Exceptionally well studied protein 14 TMDs

Question 61

2) How is UapA expressed?

Answer 61

Work with wild type form to obtain stable protein for structural analysis Express in S. cerevisiae as a fusion with GFP Functional assay available No homologue in S. cerevisiae (any activity is due to UapA) SDS-PAGE analysis: at 20 degrees protein is degrading (very unstable)

Question 62

3) Screening for mutants of UapA

Answer 62

UapA WT attached to GFP Screening method: crude extract in detergent, then carry out size exclusion chromatography Peaks: UapA in monodispersed state, aggregates and GFP At 50 degrees: denaturation of protein Single point mutation of Glycine 411 to Valine = Recovery of monodispersed protein and no free GFP Protein looses it's transport function leading to increased stability

Question 63

4) Structure of UapA monomer

Answer 63

2 distinct domains Domain moves through the membrane along with the substrate Inward facing: due to single point mutation of Glycine to Valine Bound xanthine Disulphide bridge Dimer Dimerization important for function Specificity of transporter Transporter cycle