Protein Purification and Analysis

Question 1

What are the 4 levels of protein structure?

Answer 1

Primary, secondary, tertiary, quaternary

Question 2

Define primary structure of a protein

Answer 2

Linear amino acid sequence of the polypeptide

Question 3

Define secondary structure of a protein

Answer 3

Local structure of linear segments of the polypeptide backbone atoms

Question 4

Define tertiary structure of a protein

Answer 4

Three-dimensional arrangement of all atoms

Question 5

Define quaternary structure of a protein

Answer 5

Arrangement of separate polypeptide chains into the functional protein

Question 6

Why do we purify proteins?

Answer 6

To understand…
Which proteins are involved in the process?
What does each protein do?
How does it do it?
Can we interfere with its function (therapeutics)?
Because functional studies are difficult in cells

Question 7

To study a protein in vitro, what do we need large amounts of?

Answer 7

Natural sources (tissues, bacteria, plants)
Recombinant protein expression (through cloning)

Question 8

Which organisms can we clone proteins into to obtain overexpressed proteins?

Answer 8

Bacteria (E. coli)
Insect cells (Baculovirus)
Mammalian cells
Yeast

Question 9

In basic terms, how do we purify a protein?

Answer 9

Kill the organism, lyse the cells, purify the protein

Question 10

What key molecule has been investigated using pure protein?

Answer 10

The making of ATP synthase

Question 11

Does colocalisation of proteins mean interaction?

Answer 11

Not necessarily

Question 12

What methods can we use to look at the structural biology of proteins?

Answer 12

X-ray crystallography

Nuclear magnetic resonance (NMR)

Question 13

What can we do with pure proteins?

Answer 13

Look at the structural biology

Carry out binding/enzymatic/functional assays

Question 14

What is mass spectrometry?

Answer 14

A very accurate method of molecular weight determination and identification of proteins

Question 15

What does a Western blot require?

Answer 15

An antibody specific for the protein of interest or for the affinity tag

Question 16

Briefly outline the steps used to carry out a Western blot analysis

Answer 16

1. Run an SDS-PAGE gel
2. Transfer to nitrocellulose
3. Incubate with specific antibodies
4. Autoradiography
5. Western blot

Question 17

What is SDS?

Answer 17

A detergent which binds proteins non-specifically

Question 18

What does SDS stand for?

Answer 18

Sodium dodecyl sulphate

Question 19

What does SDS do to proteins?

Answer 19

It linearises them and coats them in a negative charge

Question 20

What does PAGE stand for?

Answer 20

Polyacrylamide gel electrophoresis

Question 21

Why is it necessary for the SDS-PAGE step when purifying proteins?

Answer 21

To separate proteins by their mass

Question 22

What are absorbance measures for detecting proteins useful for?

Answer 22

Quickly screening which purification fraction contains proteins

Question 23

List some direct ways of detecting a protein of interest in a mixture

Answer 23

Absorbance
SDS-PAGE
Western blot
Mass spectrometry

Question 24

List some indirect ways of detecting a protein of interest in a mixture

Answer 24

Activity assay

Using specific properties of the protein

Question 25

What is GST?

Answer 25

A protein of 25kDa that binds Glutathione with high affinity

Question 26

How do you purify proteins using GST affinity chromatography?

Answer 26

Elute protein with a gradient of free reduced Glutathione which competes with immobilised Glutathione for binding GST

Question 27

What is FLAG?

Answer 27

A small peptide that binds specifically to Flag antibody with high affinity

Question 28

How do you purify proteins using FLAG affinity chromatography?

Answer 28

Elute protein containing a Flag tag with free FLAG peptide

Question 29

Many different types of affinity chromatography tags have been developed and are commercially available, give examples of the most common ones and what they bind to

Answer 29

Hexa-histidine (His6) binds to nickel-NTA GST binds to glutathione FLAG binds to an anti-Flag antibody

Question 30

How do you purify proteins using hexa-histidine affinity chromatography?

Answer 30

Elute protein with a gradient of Imidazole which competes with histidine for Ni2+ binding

Question 31

What is the matrix made up of in hexa-histidine affinity chromatography?

Answer 31

Ni-NTA agarose

Question 32

What do histidine amino acids have a strong affinity for?

Answer 32

Ni2+

Question 33

Nowadays, how are the majority of proteins expressed?

Answer 33

Recombinantly

Question 34

What is the most common method of protein purification considering that the majority of proteins are expressed recombinantly?

Answer 34

Affinity chromatography

Question 35

What does the level of purity achieved from affinity chromatography depend on?

Answer 35

The tag and protein of interest

Question 36

It is often necessary to do a 2nd purification step when purifying proteins using affinity chromatography, give examples of these 2nd step purification methods

Answer 36

Ion exchange chromatography | Size exclusion chromatography

Question 37

What method is only possible when using recombinant protein expression?

Answer 37

Affinity chromatography

Question 38

What do many vectors contain that is useful for affinity chromatography?

Answer 38

An affinity tag

Question 39

How is the protein released in hydrophobic interaction chromatography (HIC)?

Answer 39

By lowering the ionic strength

Question 40

What does the matrix in HIC consist of?

Answer 40

Beads of hydrophobic polymers

Question 41

How are proteins separated in HIC?

Answer 41

By their hydrophobicity (solubility)

Question 42

In HIC, what do the hydrophobic polymer beads bind?

Answer 42

Hydrophobic amino acids of proteins at high ionic strength

Question 43

What is the purification of proteins based on?

Answer 43

The physical properties of them, e.g. molecular weight, isoelectric point, solubility

Question 44

One purification methods is sufficient to obtain a pure sample of protein, true or false?

Answer 44

False, generally need to combine many purification methods such as ion exchange and size exclusion chromatography

Question 45

What does a protein's hydrophobicity profile depend on?

Answer 45

The surrounding salt concentration

Question 46

Many amino acids are hydrophobic, but where do these amino acids tend to be located within the protein structure?

Answer 46

On the inside

Question 47

What is the range of molecular weight of proteins?

Answer 47

From 2kDa up to 3.8 million Da

Question 48

How is molecular weight expressed?

Answer 48

In Dalton (Da)

Question 49

What does a protein's molecular weight depend on?

Answer 49

The number of atoms (amino acids)

Question 50

What is 1Da equal to?

Answer 50

~1g/mol | ~1.66 E-27 kg

Question 51

At a protein's isoelectric point, what is the number of positive charges equal to?

Answer 51

The number of negative charges

Question 52

At pH values below the isoelectric point, is the protein positive or negatively charged?

Answer 52

Positively charged

Question 53

Define isoelectric point (pI)?

Answer 53

The pH at which the protein's net charge is 0

Question 54

What is the charge on an amino acid at neutral pH?

Answer 54

Some amino acids are negatively charged, some are positively charged

Question 55

What do the physical properties of proteins depend on?

Answer 55

The primary sequence

Question 56

What does the choice of a protein expression system depend on?

Answer 56

The properties of the target protein | The type of analysis post-purification

Question 57

What do genes encode?

Answer 57

Proteins

Question 58

What are proteins crucial for?

Answer 58

Cellular functions

Question 59

How many genes do humans have?

Answer 59

~22,000

Question 60

How many proteins do humans have?

Answer 60

~100,000

Question 61

Define polypeptides

Answer 61

Linear chains of amino acids which fold into a specific structure depending on the sequence

Question 62

Is the backbone of an amino acid common or different in every amino acid?

Answer 62

Common to all amino acids

Question 63

Is the side chain of an amino acid common or different in every amino acid?

Answer 63

Different in every amino acid

Question 64

What is protein structure and function defined by?

Answer 64

Amino acid properties

Question 65

What types of properties can amino acids have?

Answer 65

Hydrophobic/hydrophilic Charged/uncharged Small/large

Question 66

Large molecules in size exclusion chromatography cannot enter the pores in the matrix so what happens to them?

Answer 66

They move rapidly through the column

Question 67

Small molecules in size exclusion chromatography can enter the pores in the matrix, how does this reflect the time these molecules to take to move through the column?

Answer 67

They take longer

Question 68

What is size exclusion chromatography otherwise known as?

Answer 68

Molecular sieve or gel filtration chromatography

Question 69

What is the matrix of size exclusion chromatography made up of?

Answer 69

Porous beads of carbohydrate polymer, e.g. dextran, sepharose or agarose

Question 70

What is the matrix of ion exchange chromatography made up of?

Answer 70

Beads of polymers with a charged functional group

Question 71

How does size exclusion chromatography separate proteins?

Answer 71

By their size (molecular weight)

Question 72

How does ion exchange chromatography separate proteins?

Answer 72

By isoelectric point

Question 73

What charge are anion exchange resins in ion exchange chromatography and what do they bind?

Answer 73

Positive and bind negatively charged proteins

Question 74

What charge are cation exchange resins in ion exchange chromatography and what do they bind?

Answer 74

Negative and bind positively charged proteins

Question 75

Simply, how does ion exchange chromatography work?

Answer 75

The column binds proteins with the opposite charge

Question 76

How are proteins purified in ion exchange chromatography?

Answer 76

Salt competes for ion binding sites and allow proteins to be released Typically use a gradient, e.g. 0 to 1M NaCl

Question 77

In ion exchange chromatography, what happens the greater the charge on a protein?

Answer 77

The tighter it binds the resin, the more salt is needed to dislodge it and the later it elutes

Question 78

The charge on a protein is affected by pH, and so what protein purification method is very sensitive to pH?

Answer 78

ion exchange chromatography

Question 79

What does the choice of protein purification method depend on?

Answer 79

The protein of interest: size, charges, hydrophobicity

Question 80

Before starting protein purification for analysis, what is it important to analyse first and why?

Answer 80

The amino acid sequence of the protein to help design an optimal purification stategy

Question 81

What is the maximum absorbance value of the tryptophan chemical group?

Answer 81

~280nm

Question 82

What is the maximum absorbance value of the phenylalanine chemical group?

Answer 82

~260nm

Question 83

What is the maximum absorbance value of the tyrosine chemical group?

Answer 83

~260nm

Question 84

What is the maximum absorbance value of the peptide bond chemical group?

Answer 84

~210nm

Question 85

How is absorbance used to detect proteins?

Answer 85

As a general method to use the UV absorbing properties of proteins

Question 86

At what wavelength do most proteins absorb at and what is the disadvantage to this?

Answer 86

280nm | Doesn't allow the discrimination between your protein of interest and the contaminants

Question 87

Some proteins have activities or properties that can be analysed in the test tube (in vitro), true or false?

Answer 87

True

Question 88

Do proteins have cellular functions?

Answer 88

Yes

Question 89

Anything that the protein of interest does that distinguishes it from others can be used for what?

Answer 89

An assay, i.e. a method to detect its presence

Question 90

Give examples of indirect methods for detecting proteins

Answer 90

Enzymatic activity | Detecting metalloproteins by using the properties of the metals

Question 91

When using pure proteins, you can test the direct interaction between 2 proteins using binding assays, name 2 of examples of these types of assay

Answer 91

GST pulldown | Biophysical methods

Question 92

Give an example of a functional assay

Answer 92

Phosphorylation assay