Block A Lecture 1 - From Cells to Proteins Flashcards
Why do scientists purify proteins?
As pure proteins are needed for structure analysis, sequence analysis and therapeutic preparations
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Most biological sources (such as cells) contain thousands of different proteins. What are 5 properties which proteins can be separated based on?
They can be separated based on differences in size, shape, surface charge, hydrophobicity or ligand affinity
(Slide 4)
What do resolution and yield refer to in regards to protein separation techniques?
Resolution refers to the ability to separate distinct components in a mixture whereas yield refers to the quantity of the target molecule recovered after a purification process, relative to the total amount originally present in the sample.
(Slide 4)
How are resolution and yield a balance in regards to protein separation techniques?
As methods can either give a high or low resolution or a high or low yield
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What are 5 pieces of information you should collect about a protein before starting a protein purification?
Information about the characteristics and properties of most important impurities should be collected. This includes:
Molecular weight
Isoelectric point (the pH at which a molecule has no net electrical charge)
Degree of hydrophobicity
Presence of carbohydrates
Free sulfhydryl groups (R-S-H) (disulphide bridges)
Stability (to temp, pH, organic solvents, oxygen, heavy metals, mechanical shear etc)
Proteolytic degradation
(Slide 5)
Why is information about a protein collected before protein purification?
In order to minimise the number of purification steps needed
(Slide 5)
Why would you want to reduce the number of protein purification steps?
As yield goes down the more steps you have
(Slide 6)
What occurs during the first stage of protein purification?
The protein is extracted from the source
(Slide 7)
What are 3 common examples of methods which are used to extract a protein from its source material?
Homogenisation, sonication and freeze-thawing.
(Slide 7)
What is homogenisation?
A mechanical process that physically breaks open cells by applying shear forces, achieved by devices which grind or shear cells.
The sample (cells or tissues) is forced through narrow gaps or subjected to high-speed mechanical agitation, which disrupts the cell membranes and releases cellular contents.
(Slide 7)
What is sonication?
It is when high-frequency sound waves are used to disrupt a cell. It is used for for breaking open bacterial cells, yeast, or small-scale cell suspensions.
The waves create alternating high and low-pressure cycles in the liquid, leading to the formation of microbubbles (cavitation). When these bubbles collapse, they generate shock waves that physically disrupt cell membranes.
It’s effective to deal with tough cells.
(Slide 7)
What is freeze-thawing?
Repeatedly freezing and thawing a sample to disrupt cell membranes.
When cells are frozen, ice crystals form inside and outside the cells. The formation and expansion of these crystals mechanically disrupts cell membranes. Thawing allows the cellular contents to leak out. Repeating the cycle enhances the disruption.
This method is often used for gentle cell lysis when preserving the activity of enzymes or other easily damageable molecules is important
(Slide 7)
Why are protease inhibitors included during protein purification, and why is the extract kept on ice?
Protease inhibitors are included prevent proteolytic degradation of the target proteins by endogenous proteases released from lysed cells.
The extract is kept on ice to also prevent protein degradation
(Slide 7)
What is crude cell lysate?
The unprocessed mixture obtained after breaking open cells to release their intracellular contents
(Slide 8)
Lipids and nucleic acids are components of crude cell lysate. What are 2 examples of ways each of these can be removed?
Lipids: Removed by centrifugation (the lipids will float)
Removal by adsorption
Nucleic Acids
Removal by precipitation
Addition of nucleases
(Slide 8)
What are 3 examples of methods which are used in the initial fractionation of clear lysate?
Ultrafiltration
Precipitation
Fractional Precipitation
(Slide 9)
What is ultrafiltration and what is it used for?
Liquid is subjected to pressure, which forces it through the membrane. The size of the pores in the membrane allow for the selective removal of substances based on their size and molecular weight
It has a cutoff limit for separation from 1 kDa to 300 kDa and is used for removal of salts and for concentration of the protein solution
(Slide 9)
Why would you want to concentrate a protein solution?
As it allows for a higher concentration of the target protein in a smaller volume
(Slide 9)
What is fractional precipitation, how does it work and what is it used for?
A solvent or precipitating agent is added to the mixture in a controlled manner, causing different substances to precipitate out at different stages. The process works because different substances will reach their saturation point (where they can no longer stay dissolved) at different concentrations of the precipitating agent.
It is used to remove bulk proteins and other contaminants from a sample through separating them by solubility, either precipitating them out while leaving the target protein in the sample, or the other way around. This in turn concentrates the solution.
(Slide 9)
Why is centrifugation done after fractional precipitation, and what techniques can occur after this?
In order to separate proteins which have been precipitated using a precipitation agent from the liquid which remains in the sample.
After this either the supernatant (if your protein of interest hasn’t precipitated yet) or the pellet (if it has)
If supernatant is kept, a further precipitation or column chromatography can be used to further isolate the protein of interest
Note: If pellet is kept, the same techniques can be done by the pellet must first be resuspended in a suitable buffer
(Slide 9)
What is the difference between fractional precipitation and just precipitating proteins normally, and what does this tell us about fractional precipitation?
They are both done using a precipitating agent, but fractional precipitation only adds enough to precipitate specific proteins, whereas normal precipitation adds enough to precipitate all proteins.
Fractional precipitation in turn is more controlled, and isolates specific proteins, which can make things easier down the line
(Slide 9)
Why would you ever want to use normal precipitation over fractional precipitation?
It is faster, cheaper and can be used as an initial step, or if you don’t need high selectivity.
(Slide 9)
Why are most proteins soluble?
As there are charged groups on their surface which are solvated by water
(Slide 10)
What can happen if a protein’s ability to interact with water decreases?
It can lead to decreased solubility and precipitation occurring due to protein aggregation.
(Slide 10)
