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MBB 222 > Lecture 7A > Flashcards

Lecture 7A Flashcards

1
Q

How do we isolate a protein of interest?

A

Recombinant expression systems, or protein can be purified from tissues or whole organisms:

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2
Q

Give an example of what proteins can be purified from tissues or whole organisms:

A

hemoglobin from heart tissue, pectin from plants

the tissue must be physically broken up (homogenized)

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3
Q

What is recombinant expression systems?

A

“over-express” the protein of interest in bacterial, yeast or eukaryotic cells

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4
Q

What happens in recombinant expression systems?

A
  • the gene encoding the protein is amplified by polymerase chain reaction (PCR) and inserted (cloned) into a “plasmid” or “vector”
  • the plasmid/vector also contains a selectable marker gene, e.g., bla encodes -lactamase, which destroys - lactam antibiotics – only bacteria (usually E. coli) carrying the plasmid will grow in the presence of -lactamase
  • the gene encoding the protein of interest is under the control of an “inducible” promoter to allow overexpression of that gene product
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5
Q

What are “plasmids”

A

small circular double-stranded DNA elements

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6
Q

What do both methods of isolating a protein of interest (recombinant expression systems & purification from tissue or whole organism) have in common?

A

Overexpressed protein must be isolated/purified form thousands of other proteins in cells

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7
Q

How is crude (impure) protein first isolated?

A

From the cell fraction in which it is present in the highest conc: cytoplasm, membranes, cell surface, secreted into the supernatant, organelles (for eukaryotic expression systems), periplasm (for bacterial expression systems).

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8
Q

For cytosolic proteins, the cells must be what first?

A

lysed (ruptured) by mechanical or chemical means to release cytosolic contents using ultrasonic vibration, homogenization, extrusion through a small orifice, osmotic shock, membrane-solubilisation using detergent

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9
Q

For proteins that are membrane-bound or associated with a particular organelle, must be first what?

A

Must “fractionate” the cells and purify the organelle or the membrane fraction of interest

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10
Q

Where are secreted proteins released?

A

Secreted proteins are released into the culture supernatant

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11
Q

What surface structures can be sheared off the cells?

A

Some surface structures like bacterial pili can be sheared off the cells

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12
Q

What is cell homogenate?

A

The products of cell lysis, inc membranes and organelles

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13
Q

What is differential centrifugation? What happens in it?

A

(note first lyse the cells then centrifuge)

  • Using centrifuge at increasing force to separate cells into components
  • the smaller the subcellular component the greater the centrifugal force (g-force, xg) required to sediment it
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14
Q

What does the supernatant contain after a high-speed centrifugation?

A

Enriched with hundreds of soluble proteins

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15
Q

How is the desired protein separated from the supernatant?

A

Is separated from the others based on properties of size, solubility, charge and/or specific binding affinity

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16
Q

At each step of the purification of soluble protein from the supernatant, the protein concentration and “specific activity” are assessed to ensure ____?

A

That the protein is being enriched (purified)

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17
Q

Define filtration

A

to remove large aggregates, un-lysed cells

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18
Q

Define salting out

A

Salting out of proteins (aggregation or precipitation) means crude protein purification using differential solubility

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19
Q

Define dialysis

A

to remove salts, small proteins

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20
Q

Define column chromatography

A

size exclusion, ion- exchange, affinity

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21
Q

Are most proteins are less or more soluble in high salt concentrations? Will precipitate out or in solution?

A

Less soluble in high salt conc & will precipitate out of sln

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22
Q

What compounds used to precipitate proteins?

A

Ammonium sulfate (AS) or trichloroacetic acid (TCA)

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23
Q

How can you use salt to purify crude protein?

A

A protein can be crudely purified from a complex mixture by gradually increasing the concentration of salt. Different proteins will become insoluble at different salt concentrations and can be removed from the solution by centrifugation

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24
Q

3 steps to salting out?

A
  1. salt is added at a concentration below the precipitation point of the protein of interest to precipitate out unwanted proteins for removal
  2. these insoluble proteins are “pelleted”
    by centrifugation
  3. the salt concentration of the supernatant is then increased slightly to precipitate out the desired protein - the precipitate is then resuspended (solubilized) in a physiological buffer.
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25
Q

Is salting out trial and error?

A

Yes, usually start with 10% ammonium sulfate, pellet out insoluble proteins and save them for analysis; increase the concentration to 20%, etc. Then use SDS-PAGE (see later) to identify which fraction(s) contain the protein of interest.

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26
Q

How can salts and small proteins be removed from the protein solution by dialysis?

A
  • large molecules like proteins are retained in the dialysis bag whereas small molecules are free to diffuse out into the dialysis buffer -> equilibrium
  • dialysis membranes can have different molecular weight cut-offs to allow release of different sized compounds, e.g., a 6-8 kDa membrane will allow anything smaller than ~8 kDa to pass through its pores
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27
Q

Why is the dialysis bag changed at least once?

A
  • the dialysis buffer is changed at least once to dilute out the unwanted small molecules
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28
Q

What does column chromatography allow?

A

separation of proteins based on specific characteristics like size, net charge, binding affinity, etc

(size exclusion
hydrohobic interaction
ion-exchange
affinity)

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29
Q

Define stationary phase

A

of the column: gel matrix - a porous solid material (plastic, glass, carbohydrate polymer such as cellulose or agarose, gel, bead) with specific chemical properties (charge, functional groups) and/or a specific pore size

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30
Q

Define mobile phase

A

buffered solution carrying the crude protein preparation, which may already be enriched for the protein of interest
Proteins bind the column, or migrate through the column at different rates. This depends on the properties of the proteins and the properties of the stationary phase. Proteins become separated and can then be “eluted” from the column and collected in different “fractions”.

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31
Q

The shorter or taller the column would give better resolution (ability to resolve or separate different proteins)?

A

Taller

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32
Q

Another name for size exclusion chromatography

A

also called gel filtration chromatography

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33
Q

How does Size exclusion chromatography work?

A
  • mixture of proteins in a small volume is applied to a column filled with porous beads
  • proteins are separated based on size - large proteins elute off the column first, followed by smaller ones
  • the large proteins cannot enter the pores in the polymer matrix/beads so they pass rapidly through the column
  • the small proteins enter the beads and are retarded (slowed down) and emerge from the column last
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34
Q

When fractions are collected from the column in size exclusion chromatography, what is removed first?

A

the first fractions to come off the column will have the larger proteins

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35
Q

What is ion-exchange chromatography based on?

A

Separated based on sign and magnitude of net electric charge at given pH

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36
Q

In ion-exchange chromatography, what are charged functional groups (e.g., CM, DEAE) are covalently attached to

A

cellulose or agarose beads - the stationary phase

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37
Q

In Ion-exchange chromatography, proteins with a net (+) charge will bind to what?

A

to negatively charged (e.g., CM) beads - cation exchange

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38
Q

In Ion-exchange chromatography, proteins with a net (-) charge will bind to what?

A

positively- charged beads (e.g., DEAE) - anion exchange

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39
Q

In Ion-exchange chromatography, the charge of the protein will depend on what?

A

On the pH of the buffer - pH can be adjusted to increase the net charge

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40
Q

Is CM (carboxymethyl) group + or - charged

A

negatively charged beads

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41
Q

Is DEAD (diethylaminoethyl) group + or - charged

A

positively charged beads

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42
Q

Bound proteins are eluted with increasing what concentration

A

bound proteins are eluted with increasing SALT concentration (negative and positive ions, which compete with the functional groups for binding to the protein. )

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43
Q

How does affinity chromatography work?

A

proteins are captured by a specific functional group attached to a resin or agarose bead; the protein of interest binds with high affinity to the functional group

44
Q

What are some possible functional groups and what protein do they bind to?

A

a sugar, e.g., glucose to capture/purify concanavalin A; specific DNA sequence to capture a transcription factor; substrate to capture an enzyme; antibody to capture antigen

45
Q

Can recombinant proteins use affinity chromatography? How so?

A

recombinant proteins can expressed as fusions with “affinity tags” that can be captured by a functional group or antibody or other binding protein a be fused to a protein that has a high affinity for a specific group proteins

46
Q

Name some recombinant protein examples and what they have have affinity for

A

e. g., a fusion with the protein glutathione S-transferase (GST) will bind to glutathione – the desired protein is expressed recombinantly as a GST fusion and the cell lysate is run over a gluathione-resin column to capture it
e. g., a fusion protein with a hexahistidine/polyhistidine (His6) tag binds a Ni2+-NTA (nitrotriacetic acid) column and is eluted with imidazole
e. g., an epitope tag (e.g., myc or HA or FLAG) can be recognized by a particular antibody

47
Q

How can the desired protein/recombinant protein be eluted off the column?

A
  • with a soluble competitor, e.g., glucose, DNA, imidazole, maltose
  • altering buffer conditions to reduce binding affinity (change in pH, [salt], etc.) or by adding non- immobilized functional groups (e.g., free imidazole, glucose, glutathione)
    fusion proteins can incorporate a protease cleavage site to remove the tag during or after purification
48
Q

What are antibodies (Ab) and what do they have high affinity to?

A

Proteins that have high affinity to antigens

49
Q

What are antigens

A

Protein or polysaccharides (sugar) or small molecule that antibodies bind to. They are the protein of interest, we use antibodies to pull them down

50
Q

The precise site on the antigen that the antibody binds is called?

A

The epitope

51
Q

How can antibodies be produced in lab animals?

A

by immunizing the animal with the antigen of interest.

52
Q

Downfall of producing antibodies from lab animals? Alternative?

A

expensive so sometimes an epitope for which antibodies are commercially available is engineered as an affinity tag onto the protein of interest - eg. Flag tag, Strep tag, hemagglutinin (HA) tag – these are short peptides tags that are recognized by anti-FLAG tag Ab,
anti-Strep-tag Ab, and anti-HA Ab,
respectively. These Abs can be purchased from biotech companies.

53
Q

How can total protein concentrations be determined in each fraction?

A

From the UV absorbance spectra

54
Q

What does the UV absorbance at A280 correlates with

A

Total protein concentration

55
Q

What absorption can be used to determine the protein concentration for purified fractions using the molar extinction coefficient, for that protein?

A

A280

56
Q

What does extinction coefficient depend on?

A

on the amino acid sequence of the protein (the number of tryptophan and tyrosine

57
Q

What does A80 represent when assaying/identifying proteins in different fractions?

A

represents all proteins in the solution (more accurately it will present the total concentration of Trp and Tyr),

58
Q

What happens to the A80 when the desired protein is not pure?

A

this measurement will not give an accurate concentration of the desired protein if it is not pure and has a lot of other protein contaminants present.

59
Q

Can u identify individual proteins using A80?

A

No

60
Q

The progress of protein purification, yield and purity of proteins can be assessed for each fraction or step in the purification protocol by?

A

Functional assays (e.g., enzyme activity or binding affinity) and by visualizing the proteins in using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), which separates and stains the proteins present at each step of purification.

61
Q

How to assess each fraction with SDS page?

A
  • proteins are mixed with a sample buffer that contains SDS - proteins are denatured and acquire a net negative charge
     -ME in the buffer reduces any disulfide bonds. The denatured samples are applied to wells in a gel, which is sandwiched between two glass plates and immersed in buffer. A voltage is applied across the gel. The negatively-charged proteins migrate toward the positive terminal (anode) according to size, with t`he smallest ones moving fastest – size separation.
62
Q

What is SDS

A

SDS = sodium dodecyl sulfate

an ionic detergent that denatures (unfolds) proteins

63
Q

Is SDS hydrophobic or hydrophilic?

A

is “amphipathic” has a charged end and a hydrophobic end – the long hydrophobic hydrocarbon tail associates with/inserts into hydrophobic regions on a protein and disrupts the native structure

64
Q

How does SDS bind to proteins? What charge does it give proteins?

A

SDS binds to proteins via its hydrocarbon tail and gives proteins an overall negative charge due to its SO3 group

65
Q

What does the neg charge

A

the negative charge means that proteins migrate to the anode (+ terminal) in a gel

66
Q

What slows down the migration of protein?

A

acrylamide cross-linker in the gel slows the migration of proteins – the larger the protein the slower the migration

67
Q

What is B-ME included in the sample buffer?

A

It is a reducing agent used to reduce disulphide bonds

68
Q

What is the gel used when assaying with SDS?

A
  • polyacrylamide gels made of cross-linked polymers act as “molecular sieves”
  • smaller proteins migrate more quickly (closer to bottom aka anode side) than large proteins (closer to top aka cathode)
  • note this is opposite of size exclusion chromatography);
69
Q

What is the gel stained with after?

A

dye to visualize the protein “bands” – the dye stains the proteins

70
Q

What can be calculated from this?

A

the mass of each protein (in kDa) can be calculated from those of standard proteins of known mass

71
Q

Give an example of a purification scheme for amylase

A

Homogenize E. coli cells expressing the enzyme amylase  pellet cell debris by centrifugation (discard debris).
Precipitate protein in homogenate (supernatant) using ammonium sulfate  centrifuge.
Resuspend protein pellet and run on an anion exchange column  elute.
Run eluate on a size exclusion column.
Run fraction having the correct mass on an -cyclodextrin affinity column  elute.
Measure enzyme activity of amylase, protein concentration, calculate “specific activity” (activity/mg protein) for each purification step.

72
Q

In 2D gel electrophoresis, what is the 1st dimension?

A

separate proteins according to their natural charge – isoelectric focusing.

73
Q

In 2D gel electrophoresis, what is the 2nd dimension?

A

separate the proteins according to size (SDS-PAGE).

74
Q

How many proteins can be separated in one experiment?

A

Can resolve (separate) up to 5000 proteins in one experiment

75
Q

How to conduct 2D electrophoresis?

A

Run mixture of proteins to a single lane gel strip or gel column with a pH gradient – one end has a high pH, the other end a low pH – apply a voltage across the gel – instead of migrating according to size as in an SDS gel, proteins will migrate to the point in the pH gradient where their net charge is zero – this is their isoelectric point (pI).

76
Q

What is 2D electrophoresis?

A

isoelectric focusing followed by SDS-PAGE

77
Q

What is a protein’s isoelectric point (pl)?

A

The pH value at where the protein has a net charge of zero

78
Q

Is SDS-PAGE (1D, 2D) is an analytical technique or a preparative technique

A

SDS-PAGE (1D, 2D) is an analytical technique to separate and visualize protein. The protein are reduced and denatured, and only very small amounts (micrograms) are loaded onto the gels, so they normally cannot be used further, except for mass spectrometry analysis (see next lecture), which does not require proteins to be folded and functional

79
Q

Benefit to mass spectrometry?

A

requires only very small amounts of protein, does not require that they be in their native form, and provides extremely precise mass data.

80
Q

After SDS-PAGE what process can be used?

A

Mass spectrometry

81
Q

What does mass spectroscopy allow you to determine?

A

determine the precise mass of proteins, identify proteins, sequence peptides
- enables high-throughput identification of proteins - appropriate for proteomics studies
– can analyze a complex solution of many proteins and will show peaks for precise mass of each protein present – can identify the presence of a protein by mass alone if you know the mass

82
Q

What machines are used in mass spectroscopy?

A

MALDI-TOF (matrix-assisted laser desorption ionization - time- of-flight) or ESI (electrospray ionization) spectroscopy are used

83
Q

In MALDI-TOF mass spec, what happens?

A

in MALDI-TOF mass spec proteins are ionized to gas by a laser and accelerated through a vacuum by an electric field - the time it takes to reach the detector (“time-of-flight”) is determined by their mass and charge

84
Q

When using MALDI-TOF to measure protein mass, what factors are used to separate them?

A

proteins move in the electric field according to their mass/charge ratio
smaller proteins move more quickly than large proteins with the same charge
by timing the arrival at the detector (the time-of-flight) one can measure their mass
incredibly accurate - error of 1/10,000 mass units (Daltons) for a molecule the size of myoglobin
(16.7 kDa)
can identify amino acid changes, post-translational modifications, proteolysis

85
Q

What is proteomics?

A

the large-scale study of proteins, particularly their structures and functions

86
Q

What is the proteome?

A

the study of the entire set of proteins for an organism
- usually refers to the study of the entire set of proteins for an organism (the proteome) or the set of proteins present in a particular cell type under a particular set of conditions

87
Q

What are some tools used for proteomics?

A

isoelectric focusing, protein arrays and chips, co- immunoprecipitation, mass spectrometry

88
Q

Describe a typical proteomics approach?

A

Sample -> prep steps -> 1D PAGE 2D PAGE -> cut protein spots out of gel -> digest with protease (trypsin -> Mass spec -> ESI LC-MS/MS -> MALDI MS -> Protein ID & Informatics

Basically identifies all proteins in a given sample

89
Q

What can antibodies be used for?

A

can be used to identify proteins and their locations in the cell, to purify proteins, quantify them, and to identify protein binding partners

90
Q

Describe antibody structure

A
  • made up of 4 chains: 2 “heavy chains” (blue) and 2 “light chains” (red)
  • each heavy chain and light chain pair form 2 identical antigen-binding sites
  • Fab domain - antigen-binding fragment
  • Fc domain - binds to receptors on host cells (crystallizable fragment)
91
Q

What are each heavy chain and light chain in an antibody held together by?

A
  • chains are held together by disulfide bonds and by non-covalent interactions
92
Q

What does the antigen binding site in the Fab domain bind to?

A

Binds to an epitope - a specific site on an antigen.

93
Q

Antibodies bind antigens due to what?

A

stereochemical complementarity between the antigen binding site and the epitope on the antigen. (“stereo” = shape)

94
Q

One B-cell type makes how many anti-body types?

A

one antibody type (may all be directed toward the same antigen but different epitopes)

95
Q

immune repertoire of an animal consists of?

A

of millions of different antibodies, each having its own specificity

96
Q

Each different antibody is produced by its own what?

A

Plasma or B cell

97
Q

When an antibody on the surface of a B cell binds to an antigen (e.g., during an infection or immunization) what happens?

A

B cell proliferates and and makes a lot of that specific antibody. A complex antigen or pathogen can stimulate many different B cells to produce antibodies that can bind to different epitopes on that antigen or foreign agent.

98
Q

What are polyclonal Abs?

A

derived from different B cells -> a pool of distinct antibodies that recognize a particular antigen
can have different specificities for the same antigen - bind to different epitopes (or may even bind to the same epitope but in a different way)

99
Q

What are monoclonal Abs?

A

all B cells are clones of a single B cell, so all produce the same antibody with the same specificity for the antigen
are not produced naturally – can be produced in mice by injecting an “immortalized” B cell clone into the abdomen where it will propagate and produce mAbs

100
Q

What is ELISA? Applications?

A

Enzyme-Linked Immunosorbent Assay

- for protein detection and quantification

101
Q

Describe the process for ELISA

A

protein (antigen, Ag) of interest is immobilized in a well and Ab specific for this protein (the primary antibody, 1o) is applied – the purpose is to quantify the protein based on the amount of 1o Ab bound – e.g., cholera toxin antigen, rabbit anti-cholera toxin 1o Ab
bound 1º Ab is detected by a 2º Ab that binds the first (e.g., goat anti-rabbit Ab)
the 2º Ab has an enzyme attached (linked, e.g., alkaline phosphatase) that produces a colored product when it reacts with a colorless substrate (e.g., pNPP) - substrate is added and the colored product is measured by absorbance - the more antigen in the well, the higher the signal – can be used to determine if a protein is present in a sample and to quantify it

102
Q

In ELISA, the rate of colour formation is proportional to what?

A

The amount of specific antibody

103
Q

What are the applications for immunoblotting (western blots)?

A

protein detection (and quantification)

104
Q

Difference between immunoblotting and ELISA?

A

instead of applying the antigen (Ag) to the wells of an ELISA plate the proteins in the sample are first separated by SDS-PAGE, the protein bands are transferred to a polymer membrane (e.g., nitrocellulose) using an electric field (electroblotting), and then the membrane is “blotted” with 1o Ab specific for the protein of
interest, followed by a 2o Ab conjugated to an
enzyme that will react with a substrate to form a colored product (as is done in ELISAs)

105
Q

How is the blot “developed”

A

by adding substrate for the enzyme - only the protein bands that contain the antigen will appear on the blot

MBB 222 (12 decks)

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