Brainscape's Knowledge GenomeTM

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


See full index

BIOC2004 > 3 - Protein Purification > Flashcards

3 - Protein Purification Flashcards

1
Q

What are the first three things to consider when considering a purification method?

A

The source
The activity required
The yield required

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

What use of protein would require a very high purity purification?

A

Use in structural studies, particularly NMR and XRD.

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

What things might proteins need to be purified for?

A

Enzyme Activity Studies
Structural Studies
Biotechnology (eg restriction/PCR/diagnostic enzymes)
Pharmaceuticals (TPA, Insulin etc)
Industrial use (laundry proteases, food tech, waste water treatment)

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

What headings are needed in a purification table?

A 
Activity per ml (U/ml)
Total Activity (U)
Protein Concentration (mg/ml)
Total protein mass (mg)
Specific Activity (U/mg)
Yield (%)
Purification factor
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
5
Q

How is specific activity of a protein found?

A

Dividing total activity by total mass.

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

How is protein yield calculated?

A

(Latest total activity/Initial total activity) x100

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

When selecting an organism source what factors must be considered other than those that affect the protein yield and properties?

A

Use of pathogenic organisms pose safety issues.
Public perceptions of the source for consumable products.
Patenting issues/regulatory constraints.

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

What is usually used to confirm the identity of the purified protein?

A

Mass spectrometry.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
9
Q

What is the first step in harvesting protein from microbial sources?

A

Centrifuging the cells into a pellet.

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

What is produced by lysing the source cells?

A

A crude extract

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
11
Q

What are the most popular methods of cell lysis for protein extraction?

A

Sonication
French press
Ball mill

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
12
Q

What issue is posed by lying cells with no protection for the protein?

A

Lysing the cell exposes the protein to the cellular cocktail of proteases and oxidising agents that cause denaturation and loss of yield.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
13
Q

What are the contents of a lysis buffer?

A

Various stabilising agents, neutral buffers, protease inhibitors, reducing agents, chelators (EDTA) specific cofactors required by the protein.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
14
Q

What protease inhibitors are common to lysis buffers?

A

PMSF, trypsin inhibitors.

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

What reducing agents are added to a lysis buffer, and why?

A

Compounds such as DTT and BME are used to counteract the naturally oxidising conditions of the crude extract.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
16
Q

How can protein be protected from the conditions in the crude extract other than with a lysis buffer?

A

By coexpression with a molecular chaperone.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
17
Q

What extra step in crude extract preperation may be required for purification from eukaryotic cells?

A

The protein may be localised to a particular organelle, which can be separated out by ultracentrifugation to reduce the purifying steps needed.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
18
Q

What extra steps does the use of protein purification tage add to the purification, and how are they designed to facilitate this?

A

Many tags must be cleaved off after they have been used to purify the protein to prevent them from affecting the activity or structure. They are often linked to the protein by AA linker sequences recognised by specific proteases for easy cleavage.

19
Q

What are the six main purification techniques?

A 
Salting Out
Dialysis
Hydrophobic chromatography
Size exclusion (AKA gel permeation) chromatography
Affinity chromatography
Ion Exchange chromatography
20
Q

What is biospecific separation?

A

Segregation of proteins based on their biological properties such as binding, charge, size, hydrophobicity.

21
Q

What are the basic principles of ammonium sulphate precipitation?

A

Using increasing concentrations of the hydrophilic ammonium sulphate to decrease the available water available for the proteins causing the more hyrdrophobic ones to precipitate out first.

22
Q

What property does salting out separate protein based on?

A

the concentration of ammonium sulphate which will successfully outcompete a protein for its hydration shell is dependent on the proportion of surface hydrophobic patches and water affinity.

23
Q

What is used in hydrophobic chromatography?

A

Sorts by hydrophobic surface area. Long chain hydrocarbon stationary phase and polar mobile phase.

24
Q

What kind of protein tag is used with hydrophobic chromatography?

A

Poly-Alanine tags.

25
Q

What are the principles of affinity chromatography?

A

Separating proteins based on their affinity for an immobilised form of either their natural ligand or for the stationary phase’s affinity for a recombant tag on the protein.

26
Q

What kind of tags are used for affinity chromatography?

A

His tags for use with Nickel-NTA columns (IMAC)
Stationary phase antibody raised to the protein or to protein A tag.
Expression with maltose binding protein for use in immobilised maltose column.

27
Q

What are the principles of ion exchange chromatography?

A

Stationary phase resin containing large concentrations of either a cation or an anion. Proteins of the same charge of the resin elute quickly, proteins more oppositely charged are slowed by/bound to the resin.

28
Q

What ions are present in the resin in cation exchange chromatography?

A

Anions.

29
Q

What ions are present in the resin in anion exchange chromatography?

A

Cations.

30
Q

What recombinant tag can be used with Cation exchange chromatography?

A

Poly-Arginine tails.

31
Q

What are the principles of dialysis separation?

A

By placing the extract in a semi-permeable membrane container within a larger solution of dialysate formulated to produce concentration gradients only for unwanted solutes. This must be repeated many times.

32
Q

What is homologous protein production?

A

Production of proteins for harvesting by the cells that do normally produce them.

33
Q

What kind of recombinant modifications may be done on the cells involved in homologous protein production?

A

Stimulated over-expression
Site directed mutagenesis studies
Recombinant tag
Coexpression with other proteins such as chaperones
Silencing of genes that negatively effect yield (competing for translation, toxic, proteases etc)

34
Q

What are the advantages of homologous protein production?

A

Perfect processing of the protein.

Can be used to amplify normally low conc. protein such as cytokines.

35
Q

What is heterologous protein production?

A

Use of recombinant DNA technology to cause expression of a protein in a cell in which it would not normally be found for harvesting purposes.

36
Q

What is the classic example of heterologous protein production?

A

Insulin biosynthesis in E. coli for medical use.

37
Q

What are the advantages of heterologous expression?

A

Allows for use of all tagging/co/over-expression etc technologies.
Avoids issues with the original gene source such as pathogenicity or difficulty in culturing/manipulating.

38
Q

Other than with recombinant modification, how can heterologous protein expression be used in purification?

A

If the original source and the host are very different organisms the difference in the proteins produced may be used to separate them. Eg using E coli as a host for a gene from a highly thermophilic bacteria allows incorporation of heat treatment into the purification protocol.

39
Q

How can continuous culture be achieved using recombinance?

A

Expression of the target as a fusion protein, attaching it to a highly soluble host cytoplasm protein that possesses a sorting signal that directs it for secretion.

40
Q

What are inclusion bodies?

A

Non-specific aggregates of the overexpressed target protein due to high cytoplasm protein concentration and insufficient chaperone levels.

41
Q

How can inclusion bodies be used to our advantage?

A

They represent a large concentration of the target protein, and can be harvested from the bacteria by centrifugation. They are mostly pure already and are largely immune to proteolysis so can be used to get a high yield.

42
Q

What must be done to inclusion bodies in order to obtain the target protein

A

Solubilisation to release to protein and subsequent refolding. Some extra purification often required.

43
Q

What conditions/compunds might be used to solubilise inclusion bodies?

A 
Urea
Guanidium chloride
Detergents
Organic solvents
Incubation in alkaline conditions
44
Q

How are solubilised inclusion body proteins refolded?

A

In conditions specific to the protein, tailored use of buffers/redox reagents (often glutathione). Refolding catalyst enzymes such as Protein Disulphide Isomerases ma be used.

BIOC2004 (10 decks)

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