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BS2091 Biochemistry I: from genes to proteins > Tools for protein study > Flashcards

Tools for protein study Flashcards

1
Q

Define purification of a protein

A

‘Isolation of one particular protein from all others in the starting material’

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2
Q

Why purify proteins?

A

To determine the structure: x-ray crystallography, nmr
AND/OR
To determine the function: activity, interactions

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3
Q

How can we purify proteins?

A

Exploit the protein’s:
Solubility/charge
Size
Specific interactions

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4
Q

What are the steps of protein purification?

A
  1. Selection of source
  2. Cell disruption
  3. Protein stabilisation
  4. PURIFICATION
  5. Protein identification/measure of purity
  6. Protein characterisation
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5
Q

How do you select the source for protein purification?

A

Tissue/organism producing large amounts of ‘the protein’, e.g. haemoglobin - red blood cells, bacterial proteins (100 litre fermenters)
Recombinant DNA technology - proteins expressed in bacterial/eukaryotic cells, e.g. commercially lipases (washing powder), insulin

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6
Q

How do you disrupt the cells for protein purification?

A

Rupture of plasma membrane - homogenisation, sonication, French press, osmotic lysis, freeze/thaw
Subcellular fractionation

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7
Q

Outline subcellular fractionation

A

Differential centrifugation
Homogenate is centrifuged step-by-step
Denser material sediments at lower centrifugal forces

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8
Q

How do you stabilise the proteins during protein purification?

A

Temperature (4-6 degrees centigrade)
pH (7-8?)
Inhibit protease activity (protease inhibitors, e.g. PMSF)
Bacterial contamination (decrease temperature, Na azide)
Prevent oxidation (thiol reagents, e.g. dithiothreitol)
Prevent heavy metal binding (EDTA)
Solubilise membrane proteins (Triton X-100)

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9
Q

What does an excess of dithiothreitol do to disulphide-linked chains?

A

It causes the disulphide-linked chains to become separated reduced chains

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10
Q

How can we carry out the purification stage of protein purification?

A

Ammonium sulphate fractionation
Dialysis
Column chromatography

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11
Q

What is the biological basis of ammonium sulphate fractionation?

A

Solubility of most proteins decreases with increasing (salt)

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12
Q

What is the biological basis of dialysis?

A

NOT a purification stage but an important step in the purification protocol, e.g. for removing (NH4)2SO4 imidazole etc. before ion-exchange chromatography
Separation of small molecules from large, using semi-permeable membrane

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13
Q

Give the names of some types of column chromatography

A

Gel filtration
Ion exchange
Hydrophobic
Affinity

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14
Q

In a water molecule, what dipoles are on the atoms?

A

(+) dipole on the hydrogen atoms

(-) dipole on the oxygen atom

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15
Q

How does ammonium sulphate fractionation work?

A

Salt ions compete with protein for interaction with water. As (salt) increases, protein precipitates. ‘Salting out’.

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16
Q

At what wavelength is the protein elution from column chromatography detected?

A

At 280nm

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17
Q

State how gel filtration chromatography works

A

(Gel permeation/molecular exclusion)

Separates proteins according to their size

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18
Q

State how ion-exchange chromatography works

A

Separates proteins according to their NET charge
CM-cellulose = cation exchanger (cations are +ve)
DEAE-cellulose = anion exchanger (anions are -ve)

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19
Q

Explain how ion-exchange chromatography works

A

Proteins are retained by interacting with oppositely charged beads: positively charged protein binds to negatively charged bead, negatively charged protein flows through
The strength of this interaction depends on the net charge on the protein (pl, buffer)
Retained proteins are eluted by increasing he ionic strength of the buffer

20
Q

Explain how gel filtration chromatography works

A

Small molecules enter the aqueous spaces within carbohydrate polymer beads
Large molecules cannot enter beads

21
Q

State how hydrophobic interaction chromatography works

A

Separate proteins according to their hydrophobicity

22
Q

Give examples of the possible composition of beads in hydrophobic interaction chromatogaphy

A

Phenyl-Sepharose

Octyl-Sepharose

23
Q

Explain how hydrophobic interaction chromatography works

A

Protein loaded in high (salt)
Gradient of decreasing and/or increasing organic solvent (e.g. 800mM NaCl -> 500mM NaCl, 10% ethanol)
Temperature-dependent interactions (room temperature - better than 4 degrees centigrade, but possible protein denaturation?)

24
Q

State how affinity chromatography works

A

Separates proteins due to their ‘specific affinity’ for a ligand (so can be very powerful)

25
Q

Explain how affinity chromatography works

A

Washing with buffer removes proteins that are non-specifically bound
Bound protein eluted with specific ligand

26
Q

State and briefly outline the other ways you can exploit ‘affinity’ in affinity chromatography?

A

Pseudo affinity: dye columns, e.g. Cibacron blue - binds to nucleotide binding site
Fusion proteins: maltose binding protein - X-GST
His-tag proteins: histidine binds to nickel ions (eluted with imidazole buffer)

27
Q

What is high pressure liquid chromatography (HPLC)?

A

Finer grade resins run under pressure

28
Q

What is fast protein liquid chromatography (FPLC)?

A

Protein-friendly HPLC

29
Q

What are the advantages of FPLC?

A

Better resolution than conventional chromatographic set-ups
Rapid separation
Can b used with all types of chromatography

30
Q

What is the equation for yield (%)?

A

Yield (%) = (total activity) / (total activity)initial * 100

31
Q

What is the equation for purification?

A

Purification = (specific activity) / (specific activity)initial

32
Q

How do you carry out protein identification/measure of purity when purifying proteins?

A

Electrophoresis:
i) Native polyacrylamide gel electrophoresis (PAGE) - size and charge
ii) SDS PAGE - size only
iii) Isoelectric focusing -> 2D-gels
Assay (measure of activity):
e.g. enzyme activity, binding activity (specific activity - measure of purity)

33
Q

How does polyacrylamide gel electrophoresis (PAGE) work?

A 
SDS     Na+SO4- - (CH2)11CH3
Denatures protein (breaks non-covalent bonds) and binds to protein to give an overall -ve charge
34
Q

Define the isoelectric point (pl) of a protein?

A

The pH at which the protein has an overall net zero charge.

At this pH, the electrophoretic mobility will be zero.

35
Q

What is isoelectric focusing?

A

Polyampholytes in gel used to establish pH gradient before loading sample
Proteins migrate to pH = pl
(2D-electrophoresis)

36
Q

In 2-dimensional gel electrophoresis, separation happens according to…

A
  1. Isoelectric focusing: CHARGE

2. SDS-PAGE: SIZE

37
Q

How do you carry out protein characterisation when purifying proteins?

A

Molecular mass: SDS PAGE (subunit), mass spectrometry (subunit), gel filtration, sedimentation
Subunit composition: SDS PAGE (number of different sized subunits)
N-terminal sequence analysis: Edman degradation
3D structure: X-ray crystallography, nmr

38
Q

How can the molecular mass of proteins be determined?

A

Using SDS-PAGE by comparison with marker proteins of known molecular mass.
(SDS PAGE gels can be stained with Coomassie blue to visualise the proteins).

39
Q

Electrophoretic mobility n SDS-PAGE is inversely proportional to…

A

log10(molecular mass)

40
Q

Mass spectrometry is a very accurate method for detecting what?

A

The molecular mass of proteins

41
Q

What can mass spectrometry be used for?

A

Confirming the molecular mass of a purified protein
Detection of small changes in molecular mass (e.g. addition of a phosphate)
Analysing complex mixtures of proteins
etc. etc.

42
Q

What does MALDI stand for?

A

Matrix-assisted laser desorption-ionisation

43
Q

What does ESI stand for?

A

Electrospray ionisation

44
Q

How does MALDI-TOF mass spectrometry work?

A

Measures the ‘time of flight’ (TOF) of ionised, gaseous protein molecules
MALDI -> singly charged protein ions
ESI -> multiply charged protein ions

45
Q

Outline the steps of MALDI-TOF spectrometry

A
  1. Protein sample is ionised
  2. Electrical field accelerates ions
  3. Lightest ions arrive at the detector first
  4. Laser triggers a clock
46
Q

What does sedimentation velocity measure?

A

The rate of movement of a boundary between solvent cleared of protein, and that still containing protein
Requires high speeds - 100000g
Proteins 2-20S
(S = Svedberg unit = 10^-13 sec)

47
Q

What does sedimentation equilibrium measure?

A 
Protein concentration gradient at equilibrium. (Balances diffusion against sedimentation).
Carried out at lower speeds - 1000g
Can take several days
Need pure protein
More accurate

BS2091 Biochemistry I: from genes to proteins (16 decks)

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