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Bchem 411 > Part 11 47-100 > Flashcards

Part 11 47-100 Flashcards

1
Q

What are the three main sample sources

A

Recombinant sources

Biological (model) systems

Hybridoma cell lines used for production of monoclonal antibodies

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2
Q

What are recombinant sources

A

cells that are used to express a heterologous gene for overexpression of a specific protein (usually with an affinity tag)

Ex. In bacteria yeast insect mammal plant cells

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3
Q

What is codon bias

codon optimizing

Give an example

A

Codon bias is when the codon we want in the protien is not available in the recombinant host

So we make the gene that the host prefers (codon optimize) and then put that codon in the host

Ex. UCC is preferred by the host but we want UCA, keep it as UCC so that the host makes that protein

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4
Q

What is the first choice in recombinant sources

A

Bacterial cell line

Second it eukaryotic cell lines

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5
Q

How do we maximize bacterial protein expression into the soluble phase

Why would we want it in the soluble phase

A

You can change the

growth/induction temp

IPTG concentration

length of induction (overnight)

use specialized cell lines (getting rid of the proteases so that the protein of interest isn’t chopped up)

We want the protein in the solution after centrifuging it, not in the pellet, to do this the solution is the soluble faction

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6
Q

What are examples of specialized cell lines for protein expression

A

Arctic express (switch to these if the protein is not going into soluble phase in normal cell lines)

grow the cells at low temp and slow so that they become more soluble

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7
Q

What are two other examples of specialized cell lines for protein expression

A

Rosetta

BL21 DE3 codon plus cells

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8
Q

What is production of heterologous proteins limited by

What happens

A

In bacterial cells, there is a rarity of certain TRNA that are needed to make the protein

If we force high level expression of these heterologous proteins, the limited amount of rare TRNA gets used up and stalls further translation of the protein

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9
Q

What bacterial cell line can help get over the TRNA rarity dilemma

Why

A

BL21-CODON plus

They are engineered to have extra copies of genes that encode the rare TRNA that normally get used up

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10
Q

What are human cells that are used to express proteins

A

HEK293F cells

Human embryonic kidney cells

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11
Q

What is TEV

What is the tev sequence

A

A protease that cleaves the AA sequence [ENLYFQX]

It separates the linker and the protein of interest

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12
Q

Are large proteins expressed well in bacteria

A

No

That’s when we move on to express the protein in eukaryotic cells

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13
Q

What is an example of expressing a protein in a human cell culture

A

They wanted to purify TOP2 beta and alpha

Tagged them with YFP and expressed them in the HEK293F cells

They are able to see if the proteins being made in the cell by the amount of yellow showing from the YFP

They look at the same cells and stained the DNA, they saw that the dna is showing in the nucleus

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14
Q

What did they find when from the HEK293 experiment

A

Found that TOP2 is gets shuttled into the nucleus and is a topoisomerase

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15
Q

What is Sepharose

A

A modified agarose

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16
Q

How is the recombinant TOP2a isolated from the HEK293F CELLS

What do they have to keep adding during growth of the cells and why

A

The sepharose bead has a nano body attached to it

This bead+nanobody binds to the YFP on the TOP2A

While culturing the cells with YFP, they need to keep adding media because as the cells grow they use up the media

They lyse the cells and do chromatography on them

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17
Q

after the cell is lysed in the TOP2a purification what do they do

A

The lysate is poured into a chromatography matrix

They washed all the things that weren’t the protein out

The yellow looking protein (bc of YFP) stayed in the column

Then they added tev protease to the resin to cleave the protein

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18
Q

After tev cleavage of the top 2 a what happened

What does this mean

A

To see the progress of the tev cleavage, they did SDS page with the resin

This showed the YFP-TOP2A, TOP2A, and YFP

The sds cocktail separates YFP from the nanobody , which is why we see a band of that only

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19
Q

After tev cleavage of the top 2 a what happened

What does this mean

A

To see the progress of the tev cleavage, they did SDS page with the resin

This showed the YFP-TOP2A, TOP2A, and YFP

The sds cocktail separates YFP from the nanobody , which is why we see a band of that only

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20
Q

Why did it look like there was more TOP2 a than YFP in the TOP2a experiment SDS page

A

The two proteins get stained differently by coomassie blue

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21
Q

What is different in HEK293 and HEK293F

A

The F version is a fast growing variant of the original

They grow in a more massive scale and don’t need serum

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22
Q

What are biological model systems

Why do we use them

and give an example

A

When you purify the protein from an endogenous source

This is because Some activity or factor can only be found in a specific cell type under specific condition

Ex. CDK was a factor in frog Oocytes

Or purifying protein kinases in the insulin signal pathway

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23
Q

What factor drives frog oocytes into mitosis and how was this found

A

MPF

We extract the cytoplasm from a cell that’s already in m phase then inject that into another cell

That other cell goes into m phase meaning a factor was in the cytoplasm to make it into m phase

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24
Q

What type of organisms can be used as biological/model systems

A

Viruses, non recombinant bacteria, non recombinant yeast

Animal , human , plant

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25
Q

What is the primary difference in each cell type in humans

A

The metabolism of each cell and proteins they express

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26
Q

What is special about the cells of multicellular eukaryotes

What do the cells have in common with

A

They have many different cell types for diff functions

Despite being differentiated, these cells have lots of features in common (composed of the same organelles for example)

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27
Q

What is special about red blood cells

Fat/adipose cells

A

Highly differentiated (have no nucleus)

The store energy as fat so we can use it later when starving to death

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28
Q

What are the types of model cells

A

E. coli

Yeast

Arabidopsis (plant)

Human cells in a culture

Nematode

Drosophila

Mouse

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29
Q

A cell culture is mainly made of

A

Either mammalian or human cells

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30
Q

What do cells in a culture require

A

A media with: Hormones and growth factors

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31
Q

What is a primary culture

Secondary culture

Cell line

A

When cells are taken directly from the organsim

Derived from a previously made culture

Cells with genetic modifications that make them grow indefinitely (cancerous)

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32
Q

How do you get a primary culture

What is special about fat cells

A

For fat cells you digest the ECM (which holds them together) then centrifuge

They float at the top of a centrifuged solution

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33
Q

What is red media

A

This is blood serum which is very good in the media for cell cultures

Has amino acids, glucose, vitamins

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34
Q

Before protein extraction and purification what else’s should you consider

A

Your objectives for purity and quantity

The assays you’ll do to follow your target protein

The properties of the protein that can help you purify it

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35
Q

What are the requirements of purity needed to do the three types of analysis with the our protein

This is the objectives for purity and quantity part

A

For therapeutic use, purity need to be extremely high (>99%)

For x ray crystallography high 95-99

As an antigen for antibody production moderate < (or equal) 95

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36
Q

What assays could you develop to follow your target protein

A

SDS PAGE/western blot

enzyme or functional assay (like the injection of cytoplasm into oocytes)

Protein assay (Bradford)

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37
Q

Explain why the activity would increase in the nitrate reductase inhibitor assay to track the inhibitor

A

There was an increase in activity of the nitrate reductase

This meant that the inhibitor previously was bound to the reductase, keeping its activity low

but when in solution it dissociates from the reductase and increased its activity

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38
Q

Explain how they know that the inhibitor is a protein in the nitrate reductase inhibitor assay

A

If they boil the sample and the inhibitory factor disappears (precipitates out of solution)

This means that the inhibitory factor is a protein

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39
Q

Explain how they know which fraction the inhibitor is in in the nitrate reductase inhibitor assay

A

They put each fraction of the inhibitor in a vial of nitrate reductase

The activity of nitrate reductase with that fraction begins to decrease

This means the the inhibitor was in that fraction

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40
Q

Explain what the A280 solid line is in the nitrate reductase inhibitor assay

A

The general monitoring of the inhibitor protein in each fraction as it comes through the column

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41
Q

What is the first step in sample preparation

What is the purpose

A

Cell lysis to collect and extract the sample

This is to remove all the protein population we want into the soluble phase (after centifuging)

But we still keep the in vivo state of the proteins

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42
Q

What is the in vivo state of a protein

A

The natural state of it (ex. Keeps its PTM)

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43
Q

What things can you do after collecting the sample in the supernatent but before doing the chromatography column

A

Remove non protein contaminants (lipids, nucleic acids) that were in the intial biological source

Adjust the buffer composition of the sample so it’s compatible with the column (ex. Remove salt for IEC, or change pH)

Adjust the volume and total protein concentration for the next step (if it’s gel filtration you want to reduce the volume)

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44
Q

What are the challenges that need to be tackled for proper sample prep

A

The protein complexity and dynamic range

Protecting proteins from degredation

Global or complete protein extraction

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45
Q

What does the first challenge in sample prep (The protein complexity and dynamic range) mean

A

The samples are highly complex due to PTM

the abundance of different proteins in a sample can vary widely

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46
Q

Give two examples of the The protein complexity and dynamic range challenge

A

In s. Ceravisiae, the abundance of proteins ranges from less than 50 to more than 10^6 proteins per cell (wide range)

In blood serum there is 60/80mg/ml of protein but half of this is albumin and 1/4 is y-globulin (igG)

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47
Q

What is albumin

What is IgG

A

A carrier of hydrophobic molecules (fatty acids) in the blood

Involved in the immune response

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48
Q

Slide 69

A

Idk

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49
Q

Blood plasma has a ______ dynamic range of protiens

What are some of these proteins

A

Very high

Cytokines, interleukins, interferons

50
Q

Explain how MS has allowed for more proteins to be detected and extracted

A

Before, the amount low abundance/membrane associated proteins (not easily extractable) was high compared to the abundant and soluble proteins (easily detected/extractable)

But now, because of MS, the amount of detected proteins is much higher

51
Q

Explain the challenge 2 of protecting proteins from degredation

How do we fix this challenge

A

Proteases in solution could potentially cleave your sample that you want to purify (especially after the cell has been broken open)

We’ve deleted a select few proteases from the genome of bacterial cell lines

Also we’ve can add protease inhibitors to the crude cell lysate and pooled fractions from the column

52
Q

Why do we do protein purification in the cold room

A

This decreases the protease activity so it doesn’t cleave our protein

53
Q

What are common protease inhibitors

A

PMSF
EDTA
BENZAMADINE

All cheap

54
Q

Slide 73

A

Idk

55
Q

What is included in challenge 3 global protein extraction

A

Tissue vs cell homogenization

Lysis buffers

Is the protein soluble or membrane bound

Is the protein/factor in an organelle or chromatin bound

56
Q

In Tissue vs cell homogenization what can be used to physically break the cell/tissue open

What type of lysis method is it

A

Waring blender/polytron (mechanical)

Dounce homogenizer/french press (liquid homogenization)

Sonicator (sonication)

Freezer or dry ice with ethanol (freeze thaw)

Mortar and pestle (Manual grinding)

57
Q

How does a waring blender/polytron work

What is the setback

A

Blender bladed grinds and suck everything in the centre

Not good for suspension cells because they would just stay at the top and not blend

58
Q

How does a french press work

How does a source homogenizer work

A

Liquid with the cells, the French press pushes the liquid through a small opening, the cells get busted open bc of pressure

Liquid with the cells, dounce homogenizer has a piston that pushes them down, small distance between the glass and position causes them to pop open when pushed

59
Q

How does a sonicator work

A

Put the tip of machine in the solution

The vibration from high frequency sound waves bust the cells open

60
Q

How does a sonicator work

A

Put the tip of machine in the solution

The vibration from high frequency sound waves bust the cells open

61
Q

How does a freezer with dry ice or ethanol work

What’s the setback

A

Repeated freeze thaw cycles disrupts the cells due to ice crystals forming

Not as efficient

62
Q

What buffers can be added to aid in cell lysis (for tissue/cell homogenization in challenge 3

A

Lysozyme: digests cell walls

Bead beater: glass beads move vigorously to crush cell walls

DNase/RNase : to get rid of released nucleic acids that cause viscosity

Precipitation with streptomycin sulfate (to precipitate out the dna)

Protease inhibitors

63
Q

Which treatment to lyse cells is commonly used for yeast cells

A

Lysozyme/glass beads

64
Q

Is DNASE/RNASE needed for cells that have been sonicated

A

No, this is because sonicetion also breaks the chromosomes

65
Q

What needs to be in the lysis buffer for sample prep
Why

A

A buffer (tris or hepes) at pH 7.5 and 25mM

(Because cell ph is 7.4, buffering capacity of 25mM is enough to keep at ph 7.5)

PMSF, benzamadine, protease inhibitor tablets

(Used as protease inhibitors to get rid of proteases)

Glycerol

(To make viscous, same environment as cell, stabilizes proteins)

EGTA (ca), EDTA

(Chelates ca2+, and other metals that might be needed for proteases)

Detergent like DTT (Thiol)

(To stop disulfide bond formation)

66
Q

What do proteases need to work

A

Metal ions in their active site

This is why we use EGTA/EDTA

67
Q

Membrane bound proteins are _____

A

Very hard to purify

68
Q

Where are ribosomes made

A

In the nucleolus

Timbit

69
Q

What do you have to do if your protein is in an organelle

A

You have to isolate that organelle ex. Mitochondria or nucleolus

70
Q

What is a method to separate mitochondria, lysosomes, and perixisomes

A

Place the resuspended cell pellet on top of a sucrose gradient

Centrifuge

The organelles stop at a point in the tube that’s equal to their own density and form bands at these points

71
Q

Why do we use sucrose in the method to separate organelles

A

The sucrose maintains the osmotic potential so that the organelles don’t explode

72
Q

What is the OMM and the IMS

A

Outer mitochondrial membrane

Inter membrane space

73
Q

Slide 80

A

Idk

74
Q

How do you isolate nuclei

A

Filter the supernatent, then Centrifuge

The nuclei goes in the pellet

Resuspend the pellet in mgCl2 and sucrose

Then place that on a sucrose cushion (this time there’s no sucrose gradient)

Then centrifuge in a swing out rotor (only the intact nuclei are dense enough to go through the sucrose cushion

75
Q

What g of centrifuge pellets the nuclei best

A

600

76
Q

What are the things to consider from the point of lysing cells, things to do between columns, to the final product

A

Homogenization

Clarification/centrifugation

Buffer exchange/desalting

Concentration/volume reduction

Sample cleanup

77
Q

What are the things to consider from the point of lysing cells, things to do between columns, to the final product

A

Homogenization

Clarification/centrifugation

Buffer exchange/desalting

Concentration/volume reduction

Sample cleanup

78
Q

What does the clarification/centrifugation step mean for protein sample perp and handling

A

Removing cell debris/aggresgated protiens from the sample to avoid clogging the filters or columns in future steps

Done via centrifugation

79
Q

Where is each thing after centrifugation

What is the best centrifugation speed and time for this

A

The soluble proteins are in the supernatent

The cell debris/aggregated proteins are in the pellet

Force of 15,000 g for 15 min

80
Q

Why would you need to do buffer exchange/desalting

How can you do it

A

Before doing chromatography steps, the ph may need to be changed or the salt may need to be removed

This can be done by

Gel filtration (ph change)

Dialysis (if sample is large, the small salt molecules escape out, also the ph of the sample changes to the ph of the dialysis solution)

Concentrate the samples using vivaspin or stirred concentrators (can also use to change the pH)

Precipiatation: PEG and ammonium sulfate precipitation to precipitate the protein

81
Q

How does a vivaspin concentrator work

A

The tube has a membrane separating the sample at the top from the lower compartment

The membrane has a MW cutoff (diff cutoff sizes for diff tubes 3,10,30, 100kda)

You centrifuge this and anything less than the sample cutoff goes through the membrane

sample at the top gets more concentrated sample at the bottom less

82
Q

How does a vivaspin concentrator work

A

The tube has a membrane separating the sample at the top from the lower compartment

The membrane has a MW cutoff (diff cutoff sizes for diff tubes 3,10,30, 100kda)

You centrifuge this and anything less than the sample cutoff goes through the membrane

sample at the top gets more concentrated sample at the bottom less

83
Q

How do you do do concentration/volume reduction of your sample

A

Similar to buffer exhange and desalting this uses

Vivaspin concentrators/ stirred concentrators (and concentrate a sample at 5ml down to 2ml)

Precipitation: ammonium sulfate and PEG

84
Q

How do you do do concentration/volume reduction of your sample

A

Similar to buffer exhange and desalting this uses

Vivaspin concentrators/ stirred concentrators (and concentrate a sample at 5ml down to 2ml)

Precipitation: ammonium sulfate and PEG

85
Q

Explain the sample cleanup part of sample prep and handling techniques

A

We want to remove contaminants like lipids, detergents, nucleic acids from our protein solution

We do this by precipitating the proteins with ammonium sulfate or PEG

86
Q

What’s another way to remove nucleic acids during sample cleanup

A

Use nuceleases

OR streptomycin sulfate precipitation of the dna

87
Q

What is special about PEG

A

peg can be covelently bounds to lipids

It binds water molecules and keeps the lipids soluble

Used to help constipation

88
Q

What is special about PEG

A

peg can be covelently bounds to lipids

It binds water molecules and keeps the lipids soluble

89
Q

Why would we do PEG or ammonium sulphate precipitation before doing a chromatography step

A

The precipitation helps remove other non protein molecules (clean up step)

Give some purification of the protein (through ammonium sulfate cuts)

Can reduce a large volume of sample (L) to smaller volume (mL)

90
Q

What is the advantage of PEG

What happens if we do a long dialysis to remove ammonium sulfate?

A

It carries no charge, this means we don’t have to dialyze it, saves time

The proteases have a lot of time to cleave your protein during that dialysis, proteolysis happens

This is why PEG it better

91
Q

What is the ionic strength inside cells

A

<0.15M

92
Q

What is salting in

A

When you start with low salt (<0.15M) adding more salt makes the protein salinity go up

Because the salt ions shield the protein from other ions

93
Q

What is salting out

A

When you get too high ionic strength (too much salt)

the salt competes with the protein for the water molecules

The hydrophobic patches of the proteins start to interact now and the proteins precipiates out

94
Q

Why do we do ammonium sulfate “cuts”

A

During the salting out process, diff protein have diff amount of hydrophobic patches

This means the they aggregate a diff amount of salt

So we do cut to see at what percent salt the actually precipitate

95
Q

Other than providing purification and precipitation, what else can ammonium sulfate cuts help wih

A

They can help reduce the large volume of a crude extract /lystae right after busting the cells open

96
Q

After doing ammonium sulfate precipitation, what do you do to the precipitated sample

What is the exception to this

A

Can Resuspend it in the proper buffer, then dialyze the salt away

SLIDE 88

97
Q

How do you do the calculation of amount of ammonium sulfate needed in g to get to a extraction percentage

A

First know which temp your doing it at

Then find the percent you’re starting at (ex. Zero)

Go to the percent you need (ex . 30)

That value is the g/L to add (same as mg/ml)

Times that by the ml sample you have to get mg of salt to add

Convert to g by dividing by 1000

98
Q

If doing 30-60 precipitation what is the starting percent

A

30 then go to 60

99
Q

If doing 30-60 precipitation what is the starting percent

A

30 then go to 60

100
Q

Since leg comes in a range of masses, what mass do we use for PEG fractionation

A

Peg 6000

101
Q

What does 50% w/v solution of PEG mean

A

50g of peg in a final volume of 100ml

50g/100ml

102
Q

How do you do PEG fractionation

A

Start with 30-50% w/V of peg

Add slowly via gentle stirring to the sample

Let sit for 20min then centrifuge

103
Q

What is chromatography

What is liquid chromatography

A

Separation of components using the different affinities of them for the mobile phase or stationary phase

The buffer (mobile phase) passes through the chromatography beads (stationary phase)

The diff properties of the bead and be used to purifying the proteins

104
Q

The does the column volume/bed volume mean in chromatography

A

This is the volume of beads in the column

105
Q

If we ran 10 column volumes what does this mean

A

Tan the entire column volume 10 times

If column was 1ml, ran 1ml of column volume 10times

106
Q

What are the steps in chromatography

A

First equaillibrate the column but just flowing buffer through it (to keep proteins stable)

Flow the sample through it, if proteins in the sample have affinity for the matrix, it binds

If not it flows out as flowthrough

Wash the matrix (with same wash buffer) then the proteins that are still in the matrix get washed out

Then elute the protein of interest that bound to the column using a column specific method (ion is surgery change or affinity ligand)

107
Q

What is important to remember for the column size during chromatography

A

The column size (column volume) needs to be greater than the amount of sample your adding

So that all your protein can bind to the matrix

108
Q

All chromatography matrix have a ______

A

Maximum binding capacity

109
Q

What properties of a protein do we exploit during chromatography

A

Charge (IEX)

Size (SEC, gel filtration)

Hydrophobicity (HIC, HPLC, REVERSE PHASE)

Ligand binding (Affinity chromatography)

110
Q

For tagged proteins what is the general steps to purifying it

A

The capture step: AC

The intermediate: IEX

The polishing: SEC, or IEX

111
Q

For endogenous proteins what is the general steps to purifying it

A

IF USING PEG
The capture step: IEX

The intermediate: HIC

The polishing: SEC

If using ammonium sulfate

The capture step: HIC (because we can start at high salt)

The intermediate: IEX

The polishing: SEC

112
Q

IEX samples run at

A

Higher flow rates

113
Q

Sec and gel filtration works best for

A

Very small sample

This is why we usually do it last

114
Q

Sec and gel filtration works best for

A

Very small sample

This is why we usually do it last

115
Q

What is the difference in manual and automated purification

A

Manual: don’t need much training or start up time

Easy to do parallel runs for increased throughput

Automated: convenient, high resolution, documentation

116
Q

What is the difference in manual and automated purification

A

Manual: don’t need much training or start up time

Easy to do parallel runs for increased throughput

Automated: convenient, high resolution, documentation

117
Q

WHAT are AKTA chromatography systems

A

This is where the analysis is automated

The two buffers go in the pump and mixer

Inject the sample into the machine

The sample goes through the column and the absorbance, conductive and ph it automatically calculated

Normally we would measure the things

118
Q

What are cytvia and biorad

A

Companies that make these automated column machines

119
Q

What are most chromatography beads made of

A

Cross linked Agarose

Cross linked turns it into the bead

It’s a carbohydrate polysaccharide

120
Q

What is sepharose

A

The name for the cross linked agarose bead

121
Q

What are the densities of the sepharose and what does this mean

A

4-6%

Means that a 1ml column volume is more than 0.9 (90%) water

122
Q

Slide 99

A

Densities

Bchem 411 (9 decks)

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