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BIOC1007 Simona > Protein purification > Flashcards

Protein purification Flashcards

1
Q

What are the two main frameworks for protein purification?

A
  • Purification from naturally available sources (organs, tissues, cell culture…
  • Using recombinant technologies (following introduction of expression vectors encoding a protein of interest into host cells)
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2
Q

Which source/host can….

i. Give a high yield
ii. give a high yield, but is difficult to get a hold of
iii. Give a lot of material from fermentation
iv. Is often a very good source of material

A

i. Plant cells
ii. Animal cells
iii. Bacteria
iv. Yeast

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3
Q

Which measures need to be taken in a protein purification procedure when activity is essential, regarding the host?

A

The source/host has to allow correct folding and post-translational modifications when required.

This is relevant with regard to the use of recominant technologies.

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4
Q

Which measures need to be taken in a protein purification procedure when activity is essential, with regard to the steps of the procedure?

A

Taking steps to protect activity of protein by:

a) Adding stabilisers
b) Reducing the number of purification steps to the bare minimum so as to minimise activity loss
c) Working quickly at low T

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5
Q

What are the 4 general steps of protein purification?

A
  1. Choice of framework and source/host.
  2. Extraction, solubilisation and choice of solvents (for protein of interest to be intact&functional, if required)
  3. Protein separation procedures, many of which are based on chromatography
  4. Analysis of protein purity, molecular properties and functionality
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6
Q

What are the most important factors to take into account when making choices about protein sources/hosts? (6)

A
  • Characteristics of the desired protein (properties of the protein can change with the source/host)
  • Source availability
  • Yield of product
  • Safety considerations
  • Regulatory constraints and patenting issues
  • Consumer perception
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7
Q

How is a cell extract made?

A
  1. Culture cells
  2. Separate intro centrifuge pots and centrifuge at 10-15000g
  3. Take cell pellet, resuspend in extraction buffer and subjected to breakage

Cell extract!

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8
Q

What is cell breakage/homogenisation?

A

The plasma membrane is raptured and the contents are released.

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9
Q

What are the two main categories of homogenisation techniques?

A

Mechanical: liquid shear (ultrasound, agitation, pressure) and solid shear (grinding, pressure)

Non-Mechanical: _Dessication (_air, vacuum, freeze or solvent drying) or Lysis (physical [osmotic shock, freeze-thaw, pressure release], chemical [cationic and anionic detergents], enzymatic [lysozyme and related enzymes])

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10
Q

What does a homogeneate contain? (8)

A

Cell wall fragments, membrane fragments, fibrous tissue, intracellular organelles, soluble and insoluble protein, NA, carbohydrates

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11
Q

What is the difference between cytosol and cytoplasm?

A

Cytoplasm: aqueous cell contents and suspended particles/organelles.

Cytosol: portion of the cytoplasm that remains in the supernatant after centrifugation of a homogeneate at 150,000g for 1h. It contains concentrated solution of enzymes.

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12
Q

What is differential sedimentation?

A

Cell extract: contains a mixed suspension of organlles and particles of all sizes ⇒ centrifugate at low speed ⇒ P1 contains largest components ⇒ Take SN1, centrifugate at higher speed ⇒ P2 predominantly contains medium-sized particles ⇒ Take SN2, centrifugate ⇒ P3 contains the remaining small particles.

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13
Q

Name 4 ways of “dealing with the lipid” to release membrane-associated and membrane-integral proteins

A
  1. Ultrasonication ( break cells with high freq sound)
  2. Detergent solubilisation (like dissolves like)
  3. Organic solvent extraction
  4. Digestion of phospholipid

These are normally followed by centrifugation.

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14
Q

How is denaturation prevented?

A
  • Avoid extremes of pH and temperature: use buffers around neutrality, carry out initial steps on ice
  • Avoid organic solvents and chaotropic agents such as urea and guanidinium
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15
Q

What is a chaotropic agent?

A

A chaotropic agent is a molecule in water solution that can disrupt the hydrogen bonding network between water molecules

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16
Q

How is inactivation avoided? (3)

A

Inactivation is defined as the disruption of the active site of the enzyme, which is extremely reactive. To prevent this, we must

  • Avoid oxidising conditions, especially if protein contains C-C bonds (reducing agents such as DTT or BME can be added)
  • Add chelatros (EDTA, EGTA) to avoid metal ions
  • Stabilise cofactor-requiring enzymes by adding the appropriate cofactor to the extraction/purification buffers.
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17
Q

How is proteolysis avoided?

A
  • Work fast and at low T to avoid proteolytic damage
  • Add cocktails of protease inhibitors
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18
Q

True or false:

Bufferin capacity of a buffer drops dramatically 1 pH unti either side of their pKa.

A

TRUE

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19
Q

Should a buffer be used outside of its buffering capacity range?

A

Nope.

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20
Q

Name 4 commonly used buffers.

A

HEPES, Phosphate, Succinate, TRIS

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21
Q

How does dialysis work?

A

From Tutorial 6:

Dialysis facilitates the removal of small, unwanted compounds from macromolecules in solution. In this case, it facilitates the removal of ammonium sulphate from the proteins in solution.

This is done by placing the protein solution, or sample, in a dialysis tube that is enclosed by a semi-permeable membrane, and placing the tube inside a vessel containing buffer solution. The semi-permeable membrane has pores of a specific size, which will allow small molecules such as salt ions to diffuse freely towards the buffer solution following a concentration gradient, but at the same time will retain the larger molecules inside the tube. Thus, the concentration of ions in the protein solution will be effectively reduced.

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22
Q

What’s a protease inhibitors that is effective against serine proteases?

A

PMSF (phenylmethylsulphonyl fluoride)

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23
Q

What’s a protease inhibitor effective against proteases that contain metal ions?

A

Chelating agents such as EDTA.

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24
Q

What’s a protease inhibitor effective against cysteine-dependent proteases?

A

Cysteine reagents.

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25
Q

What’s a protease inhibitor effective against acid proteases?

A

Pepstatin A

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26
Q

What are th two main types of protein separation procedures?

A
  • Crude protein separation (salt precipitation)
  • Chromatography based procedures
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27
Q

What is the principle of salt precipitation?

A

From Tutorial 6:

Ammonium sulphate is one of the most widely used agents in protein separation and protein purification through the process of salting-out. This method is based on the disparities in protein solubility in aqueous solution at different concentrations of salt.

When in aqueous solution, a protein is surrounded by a hydration shell. Ammonium sulphate (and other similar agents) can be used to strip the water molecules away from the protein and eliminate the hydration shell, thus forcing the proteins to interact with each other and precipitate. Given that different proteins have different compositions, i.e. different ratios of hydrophilic and hydrophobic patches, they precipitate at different salt concentrations.

When the salt concentration is low, its presence stabilises the various charged groups on the protein, thus attracting the protein into the solution and enhancing its solubility (salting-in). However, as the salt concentration increases, a point of maximum solubility is attained, after which, assuming that the concentration of salt continues to increase, there won’t be sufficient water molecules to interact with the protein molecules (salting out). This is when the protein starts to precipitate. A diagram of the process is provided below.

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28
Q

What is the principle of separation in chromatography-based methods?

A

As proteins migrate through the column, they are retarded to different degrees by their different interactions with the matrix material. Individual types of proteins (such as A, B, and C) gradually separate from each other, forming bands and eluting separately.

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29
Q

What are the stationary phase and the mobile phase? (in general, for chromatography-based purification procedures)

A

Stationary phase- solid porous matrix

Mobile phase- solution from the reservoir and solution containing mixture of proteins (protein sample)

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30
Q

What is the separation principle behind size exclusion chromatography?

A

Gel filtration separates proteins according to their size and shape.

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31
Q

What is the separation principle behind chromatofocusing?

A

The PI of different proteins

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32
Q

What is the separation principle behind affinity chromatography?

A

It exploits a particular biological function (binding affinity).

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33
Q

How does gel filtration chromatography work?

A

The underlying principle behind this method is that different proteins vary in size and shape, and thus can be separated accordingly. A solution containing a variety of proteins, including the protein of interest, flows through a separation column. The matrix of the column contains a cross-linked polymer of beads that have pores of a certain size.

When the mixture of proteins travels through the column, the smaller proteins will be retained in the pores and will take longer to make their way through the column because they will follow a more intricate path. On the other hand, larger proteins will not be retained in the pores and thus will migrate through the column a lot faster, i.e. they will elute earlier. Different fractions can be collected and then assayed for specific enzyme activity.

Proteins whose Mr is above the exclusion limit are consequently excluded from penetrating the gel matrix.

34
Q

How does ion exchange chromatography work?

A

Specific proteins have distinct net electric charges at a given pH, due to the ionisation state of the amino acid side chains that they’re made up of. These differences in net charge between different proteins can be exploited for protein separation and purification by using ion exchange chromatography.

The matrix of the separation column for anion exchange chromatography contains positively charged particles, which locks the more negatively charged proteins to the matrix, while more positively charged proteins will have little or no affinity for the synthetic polymer in the matrix and will flow through the column.

To elute the proteins that are tightly bound to DEAE-cellulose, the protein-matrix interactions must be broken. This can be achieved by gradually increasing the concentration of competing free salt in the surrounding solution, a method that is known as elution by anionic counter ion.

35
Q

In anion exchange chromatography, the matrix is ________ charged.

A

Positively

36
Q

In cation exchange chromatography, the matrix is ________ charged.

A

Negatively.

37
Q

Above its PI value, a protein has a _____ charge.

A

Negative

38
Q

Below its PI value, a molecule has a _______ charge.

A

Positive.

39
Q

What is the underlying principle of ion exchange chromatography?

A

Separating proteins using theri net charge at a given pH:

At a given pH, proteins which have different PI valuse will have a different charge.

40
Q

What is the basis of hydrophobic interaction chromatography?

A

HIC exploits the differences in hydrophobicity of proteins. The matrix is a synthetic polymer containing bound hyrdophobic groups.

Contrary to ion-exchange, binding is at high salt, elution is at low salt: water shield hydrophobic interactions between protein and matrix, salt strips away the water and allows the interactions to take place.

More in detail:

Close to the surface of the hydrophobic ligand and solute, the water molecules are more highly ordered than in the bulk water and appear to ‘‘shield off’’ the hydrophobic ligand and the hydrophobic protein patch. Added salt interacts strongly with the water molecules leaving less water available for the ‘‘shielding off’’ effect.

41
Q

What is the basis of affinity chromatograpy, and how does it work?

A

AC separates proteins by exploiting their binding specificities, i.e their biological function.

Proteins retained on the column are those that bind specifically to a ligand, cross-linked to the beads.

Proteins that do not bind to the ligand are washed thorugh and the bound protein of interest is eluted by a solution containing free ligand or other competing small molecule.

42
Q

Which methods are used to analyse the purity, properties and functionality of a purified protein? (4)

A
  1. Measuring protein amounts/concentration
  2. Assessing protein heterogeneity
  3. Determining molecular mass
  4. Enzyme assays and other functional assays
43
Q

True or false:

Spectrophotometric methods are often used to measure protein concentration.

A

So true.

44
Q

What is the Beer-Lambert law?

A
45
Q

What is the principle of the SDS page?

A

Protein is denatured with heat. Also, thiols are added in order to remove the disulphide bonds that contribute to stabilising the protein’s tertiary structure.

Protein is treated with SDS (sodium dodecyl sulfate):SDS molecules bind to the unfolded peptide chain and give them a strong, uniform -ve charge.

This allows size to be the only separation principle, as charge is uniform.

46
Q

True or false:

SDS page allows to determine the native (non-denatured) size of the protein.

A

False, that’s size exclusion.

47
Q

How can we determine the non-denatured, native Mr of a protein?

A

By means of size exclusion chromatography:

Ve (elution volume) depends mainly on molecular size.

By comparing the Ve of a protein of interest with that of a known standard, its Mr can be determined.

48
Q

What is the general principle of MS?

A

Molecules to be analyzed are first ionized in a vacuum. When the newly charged molecules are introduced into an electric and/or magnetic field, their paths through the field are a function of their mass-to-charge ratio, m/z. This measured property of the ionized species can be used to deduce the mass (M) of the molecule with very high precision.

49
Q

What are two MS methods for analysis of proteins?

A

MALDI MS (matrix-assisted laser desorption/ionisation MS)

ESI MS (electrospray ionisation MS)

50
Q

How does ESI MS work?

A

A protein solu@on is dispersed into highly charged droplets by passage through a needle under the influence of a high-voltage electric field. The droplets evaporate, and the ions (with added protons) enter the mass spectrometer for m/z measurement.

The spectrum generated is a family of peaks, with each successive peak corresponding to a charged species increased by 1 in both mass and charge. A computer-generated transformation of this spectrum is shown in the inset.

51
Q

What are the 3 basic components of an enzyme assay?

A
  1. Reaction mixture (includes buffer, substrate, cofactors or stabilisers if required by enzymes), often brought to T of the assay prior to initiating the reaction.
  2. Initiating the incubation by addition of enzyme or fractions from a purification protocol.
  3. Some assays require termination by inactivating the enzyme, which can sometimes be combined with colour development.
52
Q

What are the units of enzyme activity?

A

micro moles/ min

Enzyme activity is defined as the amount of enzyme that catalyses the conversion of 1 micromole of substrate per minute under defined conditions of temperature and pH.

53
Q

What is activity?

A

Total units of enzyme in a solution (remains the same throughout purification)

54
Q

What is specific activity?

A

number of enzyme units per miligram of total protein (activity expressed as a ratio)

It is a measure of enzyme purity: increases throughout purification procedure, becomes maximal and constant when enzyme is pure.

55
Q

How is the final purification fold calculated?

A

e.g in a 5 step purification procedure

Overall /final purification factor (fold) = Specific Activity at step 5/Specific Activity at step 1

56
Q

How is the overall yield (%) calculated?

A

e.g in a 5 step procedure

Overall yield (%) = [Activity (total) at step 5/ Activity (total) at step 1] x 100

57
Q

What is homologous protein production?

A

The removal or isolation of a gene sequence/cDNA derived from a particular species and to subsequently produce its gene productby recombinant means in the same species.

In this case the host cell is producing a recombinant protein that it is capable of producing naturally. In these cases the manipulation may allow the product to be produced in higher quantities and problems such as gene repression can be over come. + OSDM, maybe?

58
Q

What is heterologous protein production?

A

The expression of recombinant proteins in cells which they do not naturally occur is termed heterologous expression. The vast majority of recombinant proteins are produced in this way

59
Q

Name 4 host cells

A
  1. Bacteria (E.Coli)
  2. Yeasts
  3. Insect cells

(these first three can be grown in great quantities)

  1. Mammalian cells (if protein to be purified requires post-translational modifications)
60
Q

What are expression vectors?

A

Expression vectors include a gene of interest that can be modified by adding “tags” to generate fusion-recombinant proteins
Tags for affinity purification, sometimes also solubility

e.g polyHis, GST, FLAG-peptide, MBP

61
Q

How are tags introduced?

A
62
Q

What is the main function of tags?

A

To allow for simpler purification based on one, single affinity chromatography step.

63
Q

What are the IL and what is used for elution of

a) Poly-His
b) GST
c) FLAG-peptide
d) MBP

A

a) Ni2+, Co2+ - Imidazole
b) Glutathione - reduced glutathione
c) Anti-FLAG monoclonal antibody - FLAG-peptide
d) Cross-linked amylose (maltose)-Maltose (disaccharide derived from amylose)

64
Q

What is IMAC?

A

Immobilised metal ion affinity chromatography, basis for affinity chrm with GST tag

65
Q

Explain purification with affinity chrm using GST

A
66
Q

Explain purification with affinity chrm using a His6 tag bound to MBP and recombinant TEV-protease with a His6 tag

A

MBP is only used for solubility and purifica.on is based on His6 tag (IMAC)

Recombinant TEV- protease also has His6 tag and is retained on the matrix together with cleaved His6-MBP tags.

Only cleaved target protein is unbound (in flow through = ft)

67
Q

How can tags be used for detection?

A

Antibodies can be used to recognize a tag in a recombinant fusion protein- can be used to detect the protein for example by Western blotting

68
Q

4 adv and 4 disadv of heterologous protein production in E.Coli

A
69
Q

One of the disadv of heterologous protein production in E.Coli is the formation of inclusion bodies. What are the causes?

A

  • Very high local concentrations of expressed protein in the cytoplasm lead to non-specific precipitation
  • Insufficient chaperones to aid in the correct folding with subsequent aggregation of partially folded and mis-folded intermediates
70
Q

6 ways of facilitating the production of soluble heterologous proteins in bacteria (i.e approaches to avoid the production of inclusion bodies)

A

  • Empirical determina@on of the op@mal expression system in terms of host strain, plasmid, plasmid copy number and promoter sequence etc.
  • Expression of the desired product as a fusion protein, fused to a highly soluble and na@ve host cytoplasmic protein.
  • Expression behind an export signal such as a signal sequence that directs the expressed protein to the periplasmic space or out of the cell completely.
  • Co-expression with chaperone proteins that improve the solubility of the expressed protein by aiding in the folding pathway of the protein.
  • Providing appropriate cofactors that may increase the soluble yield of a recombinant protein requiring that cofactor.
  • Growth of the recombinant cells at sub-op@mal temperatures: growth of E. Coli cells at lower temperatures than op@mal oIen discourages inclusion body forma@on.
71
Q

What are the 3 major effects that the advent of recombinant DNA technology has had on the science of protein purification?

A
  1. Overcoming problems of source availability
  2. Overcoming problems of source safety
  3. Modifications made possible
72
Q

Overcoming problems of source availability

A

Many naturally occurring proteins are expressed at low level (e.g. cytokines) or may only exist in difficult sources (e.g. human tissue).

High level of expression achieved by recombinant technology
results in high specific ac.vity in the cell extract and consequently fewer purifica.on steps and overall fold-purifica.on is needed to achieve the required level of purity.

73
Q

Overcoming problems of source safety

A

Some proteins are produced naturally by dangerous or pathogenic species (e.g. microbial pathogens, snakes etc.); there is a possibility of dangerous contaminants during purifica.on or in the final product.

Furthermore associated hazards may not be clearly defined
(you may not know what problems are associated with a par.cular .ssue).

Recombinant technology procedures are developed within strict, standardized regula.ons.

74
Q

Modifications are possible

A

target protein can be modified if desired by procedures (such as site-directed mutagenesis) which can lead to improved quali.es of the desired product and/or facilitate purifica.on (e.g. tags)

The tag itself may enhance stability and solubility of the protein.

Very large purifica.on in a single step usually results from the use of affinity chromatography based on a specific tag.

75
Q

4 examples of proteins produced recombinantlu of industrial/medical significance

A

Insulin

Glucagon

HCG

Blood factors

76
Q

3 factors that influence the acceptability of genetically engineered proteins

A
  1. benefit to user (whether or not it cures life threatening diseases)
  2. Intimacy with product
  3. May be the only source?
77
Q

What is insulin used for?

A

Treatment of type I diabetes, as the hormone is no longer produced internally. Also used in over 40% of type II diabetes patients

78
Q

adv and siadv of recombinant insulin

A
79
Q

What is chymosin? what are its adv and disadv?

A

Acid protease used to produce milk solids for cheese manufacture.

80
Q

What are the adv and disadv of recombinant proteases, lipases and amylases used as biological cleaning agents?

A

BIOC1007 Simona (5 decks)

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