Brainscape's Knowledge GenomeTM

Browse over 1 million classes created by top students, professors, publishers, and experts.


See full index

BIOC2004 CH > 2. Protein Purification & Heterologous Protein production > Flashcards

2. Protein Purification & Heterologous Protein production Flashcards

1
Q

What do we purify proteins for (5)?

A
  1. Protein/enzyme activity studies
  2. Structural/functional characterisation - X-ray crystal, NMR
  3. Pharmaceuticals - drugs nd shit
  4. Industry - enzymes nd shit (proteases for detergents)
  5. Biotechnology - restriction enzymes nd shit
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
2
Q

Describe the 3 stages of a purification protocol and the methods used to achieve this

A
  1. Cell extract preparation - cell disintegration, centrifugation, detergents
  2. Fractionation - precipitation, chromatography
  3. Diagnosis/Characterisation - activity/protein assay, electrophoresis, imumunodetection, mass spec, spectroscopy, AA sequencing
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
3
Q

What are the three types of protein chromatography?

A
  1. Ion exchange
  2. Gel filtration
  3. Affinity chromatography
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
4
Q

What are the three types of affinity chromatography?

A
  1. Biospecific
  2. Immunoaffinity
  3. Immobilised metal chelate affinity chromatography
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
5
Q

Describe the process of affinity chromatography

A

An affinity ligand (may be antibody, ATP etc) is cross linked w/ a spacer arm to an agarose build (gel matrix). The affinity ligand binds a specific protein. The protein is then eluted

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
6
Q

What is imumunodetection?

A

A protein diagnosis method involving using specific antibodies to detect the presence of a protein.

The protein is first run on a gel electrophoresis, then transferred to membrane and probed with antibodies

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
7
Q

Describe the process of IMAC

A

Immobilised Metal Chelate Affinity Chromatography.

Spacer arm cross-linked to agarose beads can coordinate nickel ion. The 3 free coordination spaces on the Ni can coordinate His residues on proteins.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
8
Q

What problems has recombinant DNA technology allowed us to overcome (in terms of proteins)?

A
  • Problems of source availability
  • problems of source safety
  • modifications!
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
9
Q

What is the difference between homologous and heterologous protein production?

A

Heterologous: expression of a gene in a host organisms which doesn’t usually contain the protein

Homologous: over expression of a gene in the same system it originates

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
10
Q

Give an example of 7 different heterologous protein expression systems

A
  • microbial cells - E. coli
  • yeast cells - S. cerevisiae
  • Fungal systems
  • Plants - Tobacco
  • Insect cells
  • Animal cells - CHO
  • Transgenic plants/ animals
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
11
Q

What are some problems with heterologous protein expression?

A
  • Lack of protein expression
  • Protein degradation
  • Poor solubility
  • Formation of inclusion bodies
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
12
Q

What may cause lack of protein expression in heterologous protein expression systems (6)?

A
  • structural changes in recombinant gene
  • codon preference
  • mRNA instability
  • incorrect post translational processing
  • coenzyme availability
  • folding dynamics
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
13
Q

Why do inclusion bodies form (4)?

A
  • High local concentration of expressed protein leading to precipitation
  • Not enough chaperones to aid in folding (leads to misfolding and aggregation)
  • Cytoplasm has a reducing environment which doesn’t allow disulphide bonds to form
  • Bacterial proteins do not undergo post translational processing which could make proteins less stable in eukaryotes
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
14
Q

How may inclusion bodies be helpful?

A

In protein purification.

Density and insolubility of proteins makes them susceptible to sedimentation at low G forces.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
15
Q

Describe the process of protein purification using inclusion bodies

A
  • Centrifugation causes sedimentation of inclusion bodies
  • IB then added to buffer and urea/guanidium chloride/detergents/high pH are used to solubilise the IB’s
  • The denaturants are then removed by dialysis/dilution/filtration
  • Protein is refolded (need correct ox/red agents, sometimes need protein disulphide isomerase - refolding catalyst)
  • choose suitable buffer to keep proteins in
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
16
Q

What approaches are used to express soluble proteins (5)?

A
  • Change expression conditions (low temp, rich media)
  • Expression of different variants of the protein (diff. N/C terminals)
  • Coexpression of interacting proteins (if in protein complex usually then coexpress w/ other complex proteins)
  • Ligand supplementation (addition of ligand)
  • Coexpression of chaperones
17
Q

What extra efforts are made to avoid inclusion body production when expressing large/complex proteins (6)?

A
  1. Optimal Expression System determined - considering host strain, plasmid, copy number, promoter sequence
  2. Fusion protein expressed - protein fused to highly soluble, native cytoplasmic protein
  3. Export signal - added to protein to direct it out of cell
  4. Chaperone co-expression - aid folding of protein
  5. Providing cofactors (or cofactor precursor)
  6. Sub-optimal conditions - growth at lower temperatures prevents inclusion body formation
18
Q

Why is purification of recombinant proteins easier then purifying non-recombinant ones (5)?

A
  1. Higher expression level of target protein
  2. Increase in ratio of target proteins to contaminants
  3. Don’t need initial concentration steps
  4. Recombinant protein can exhibit properties not shown by non-recombinant (e.g. thermostability)
  5. Inclusion bodies
19
Q

How may fusion proteins be produced for the purpose of target protein purification?

A
  • Specific peptides or protein tags are introduced to either end of protein
  • Linker sequence between protein/tag is a target for cleavage agent so tags can be easily removed
20
Q

What tags can you add to proteins (5)? What are they used for?

A
  1. Polyarginine tail - added to C terminus to help Ion exchange chromatography
  2. Polyphenilalanine tag - aids hydrophobic interact chromatography
  3. Maltose binding protein - purified on maltose affinity column
  4. Polyhistidine tag - aids metal chelate chromatography (IMAC) purification
  5. Protein A - aids immune-affinity purification w/ IgG columns
21
Q

How may tags be removed?

A

Cleavage with trypsin, chymotrypsin, enterokinase, or more specific proteases (TEV protease, precision proteases)

22
Q

What are GPCRs?

A

G protein coupled receptors:

  • Integral membrane proteins found on surface of every human cell
  • Transmit extracellular stimuli to the interior of the cell
  • regulate metabolism, neurotransmission, visual perception, immune response
  • major target for drug development but struggle struggle to heterologously express
23
Q

What are the advantages and disadvantages of using E. coli as a heterologous expression system (8)?

A

Advantages:

  • low cost
  • ease of use
  • variety of strains
  • rapid doubling time
  • uniform isotopic labelling (tracking of isotopes is consistent through cells)

Disadvantages:

  • no native G-proteins/GPCR interacting proteins
  • Don’t perform post translational modifications (some GPCR’s require glycosylation)
  • Differing lipid composition can affect ligand binding
24
Q

How is E. coli used to express GPCRs (process)?

A
  1. Signal peptide of E. coli periplasmic maltose binding protein is attached to GPCR N-terminus (targets protein to PP membrane)
  2. Expression then occurs under control of weak promoter, at 18-25deg., long time, protease lacking cells (DH5a)
  3. Glucose and ligand addition increase expression

Proteins are produced as inclusion bodies during high level GPCR production

25
Q

What are the advantages of using inclusion bodies to produce proteins in E. coli (3)?

A
  1. Expressing the GPCRs in inclusion bodies reducing the toxicity to the cells
  2. IB’s are easy to purify and resistant to proteolysis
  3. 100+mg IBs produced per litre culture

Sadly, refolding of GPCRs is required when using this method

26
Q

What are the benefits of using yeast systems to produce GPCR proteins?

A
  • Easy foreign DNA uptake (well-understood genetics)
  • Rapid screening of transformed cells
  • Mammalian-like folding (yeast have correct organelles)
  • Can perform post-translational modifications and form disulphide bonds
  • Easy to alter culture conditions to promote higher yields
  • Introduction of accessory/heat shock proteins promotes folding
27
Q

What are the problems when using yeast systems to express GPCRs?

A
  • Do not always perform post translational modifications predictably
  • Nonuniform glycosylation can cause difficulties in crystallisation (homogeneity required)
  • Difference in membrane-lipid composition affects ligand-binding activity
28
Q

What are the benefits of using baculovirus/insect cell systems to produce GPCRs?

A
  • Correct folding of recombinant protein
  • Disulphide bond formation and post-translational modifications identical to mammalian cells (apart from glycosylation)
  • Sf9 cells also express G-alpha subunits which interact with GPCR
29
Q

Where in the genome is the GPCR gene embedded in the baculovirus system? How?

A

In a nonessential region of the viral genome

This is done by homologous recombination with a transfer vector (when vector and virus are cotransfected in insect cells)

30
Q

What are the problems with baculovirus/insect cell system?

A
  • Plasma membranes not identical to mammalian cells
  • requires high-toter viral stocks to infect cultures (stock production is time-consuming)
  • can only be kept for 30 days after which there is a loss in production due to high amounts of viral particles
  • heterogenous populations of receptors due to variation of post translational modifications affects protein crystal quality
31
Q

What are the difficulties in using mammalian cells to express GPCRs?

A
  • GPCR is toxic to cell in high levels (so not reliable) - need inducible systems
  • Costly, time consuming
32
Q

How does cell-free expression work? What does it enable?

A

All components needed are in vitro, obtained through making a cell lysate

Enables expression of proteins which would usually interfere with cell physiology, side steps problems with in vivo expression (protein degradation, aggregation, loss of DNA, toxicity), easier to isotopically label

33
Q

What are the issues with cell are expression?

A
  • Costly
  • lacks GPCR interacting signalling components
  • lacks post translational modifications (low expression levels, misfolding)
  • difficult to add cellular components and liposomes
  • scale-up problems - 1ml reaction volumes

BIOC2004 CH (11 decks)

Brainscape helps you reach your goals faster, through stronger study habits.
© 2024 Bold Learning Solutions. Terms and Conditions