Protein Purification Techniques and Strategies

What are the main functions of proteins?

Proteins serve various functions including catalytic (enzymatic), storage, hormonal, defensive, transport, receptor, and structural roles.

What percentage of the dry mass of most cells is made up of proteins?

Proteins account for more than 50% of the dry mass of most cells.

What are amino acids and their significance in proteins?

Amino acids are organic molecules that serve as the building blocks of proteins, differing in properties due to their side chains (R groups).

What is a polypeptide?

A polypeptide is a polymer of amino acids linked by peptide bonds.

What defines a protein?

A protein is a biologically functional molecule that consists of one or more polypeptides.

What are the ends of a polypeptide called?

The ends of a polypeptide are referred to as the carboxyl end (C-terminus) and the amino end (N-terminus).

What are the four levels of protein structure?

The four levels of protein structure are primary, secondary, tertiary, and quaternary.

Why is protein purification important?

Protein purification is important to determine physical properties, conduct functional studies, analyze protein-protein and protein-ligand interactions, and for structural studies.

What are some methods used for structural studies of proteins?

Structural studies can involve techniques like protein crystallography and NMR structure determination.

What are the key components of protein expression systems?

Key components include genetic elements essential for expression and specific regulatory elements such as Plac and T7 expression systems.

What is a common troubleshooting step for low protein production?

Optimizing the level of inducer, time of induction, and temperature of the induction step can help address low protein production.

What is gene fusion strategy in protein purification?

Gene fusion strategy involves genetically fusing the gene encoding the target protein with a gene encoding a purification tag to facilitate specific capture during purification.

What are the four important criteria in purification strategy design?

The four criteria are monodispersity, purity, homogeneity, and yield.

How is purity measured in protein purification?

Purity is measured by electrophoresis and specified as the target protein-to-total protein ratio.

What is the significance of yield in protein purification?

Yield is significant as it indicates the total amount of protein obtained, measured by absorption at 280 nm and activity assays.

What are the common chromatography types used in protein purification?

Common chromatography types include affinity chromatography (AC), ion exchange chromatography (IEX), and hydrophobic interaction chromatography (HIC).

What is the role of high-resolution size exclusion chromatography (SEC) in purification?

High-resolution SEC is often used at the polishing stage of purification to assess the final product.

What is the purpose of using an online protein detection method in purification?

Online protein detection methods provide real-time monitoring of protein yield, often producing a chromatogram.

What is the relationship between purification steps and yield/purity?

The yield and purity of a protein can be affected by the number of purification steps taken.

What is the significance of the R group in amino acids?

The R group determines the unique properties of each amino acid, influencing the structure and function of the resulting protein.

What is the difference between a protein and a polypeptide?

A polypeptide is a chain of amino acids, while a protein is a functional molecule that may consist of one or more polypeptides.

What is the importance of structural studies in protein research?

Structural studies are crucial for understanding protein function, interactions, and for applications in drug design.

What factors can affect protein expression levels?

Factors include the level of inducer, time of induction, and temperature during the induction process.

What is the purity of the protein after the purification step?

It is determined by the ratio of specific activity of the current protein fraction to the specific activity of a previous step.

What does a higher purification factor indicate?

A higher purification factor indicates a more effective purification step.

How are mass percent solutions defined?

Mass percent solutions are defined as grams of solute per 100 grams of solution.

What is an example of a 20% by mass solution?

20 g of sodium chloride in 100 g of solution.

How are volume percent solutions defined?

Volume percent solutions are defined as milliliters of solute per 100 mL of solution.

What is an example of a 10% by volume solution?

10 mL of ethyl alcohol plus 90 mL of water making approximately 100 mL of solution.

What are mass-volume percent solutions indicated by?

They are indicated by w/v % and defined as grams of solute per 100 mL of solution.

What is an example of a 1 w/v % solution?

1 g of phenolphthalein in 100 mL of 95% ethyl alcohol.

What is the formula for molarity?

Molarity (M) = Moles of solute / Liters of solution.

What is the formula for mole fraction?

Mole Fraction (χ) = Moles of solute / Total moles of solution.

What is the formula for molality?

Molality (m) = Moles of solute / Kilograms of solvent.

What is the purpose of dilution in solution preparation?

To prepare a desired solution by adding water to a concentrate.

What are some methods for cell lysis in protein extraction?

Chemical/Osmotic Lysis and Mechanical Lysis.

What is sedimentation in the context of protein purification?

Sedimentation involves the separation of soluble proteins by centrifugation.

What are some factors that can denature proteins during cell disruption?

Heat, mechanical agitation, pH change, inorganic salts, polar organic solvents, soaps, and detergents.

What is the importance of temperature stability in protein purification?

It necessitates working rapidly at lower temperatures to prevent denaturation.

Why is pH stability important in protein purification?

It influences the selection of buffers for extraction and purification processes.

What role do organic solvents play in protein purification?

They affect the selection of conditions for reverse phase chromatography.

How does salt concentration influence protein purification?

It affects the selection of conditions for precipitation techniques and hydrophobic interaction chromatography.

What is the significance of protease sensitivity in protein purification?

It necessitates the fast removal of proteases or the addition of inhibitors.

What is the role of molecular weight in chromatographic separations?

It influences the selection of gel filtration media.

What are the four general chromatographic steps?

1. Solvent A, 2. Analyte in Solvent A, 3. Solvent B, 4. Elution with Solvent A.

What happens during the elution process in chromatography?

Solutes/proteins bound to the stationary phase are washed out with a buffer containing solvent B.

What is an automated FPLC system?

A system with an advanced level of automation used for protein purification.

What types of gels are used in electrophoresis?

Agarose and acrylamide gels.

What are the two types of acrylamide gel systems in electrophoresis?

Continuous and Discontinuous systems.

What is SDS PAGE?

A type of protein electrophoresis that separates proteins based on their size.

How does SDS PAGE work?

Proteins are boiled in sodium dodecyl sulfate and beta-mercaptoethanol, which disrupts their structure and imparts a negative charge, allowing them to migrate towards the positive pole in an electric field.

What happens to proteins during SDS PAGE?

They contain only primary structure and migrate at a rate proportional to their linear size.

What is the difference between continuous and discontinuous acrylamide gel systems?

Continuous systems use a single separating gel with the same buffer, while discontinuous systems have a stacking gel layered on top of a separating gel, providing greater resolution.

What is the benefit of using a discontinuous acrylamide gel system?

It offers much greater resolution compared to a continuous system.

What is the basis of solute separation in chromatography?

Solutes that interact more strongly with the stationary phase take longer to pass through the column.

What happens to solutes that have weak or no interactions with the stationary phase in chromatography?

They elute very quickly.

What are the types of chromatography based on solute retention mechanisms?

Gel filtration, Hydrophobic Interaction, Ion Exchange, and Affinity Chromatography.

What is the purpose of affinity chromatography?

It offers high specificity and resolution, allowing for the purification of proteins that would be difficult to isolate using other techniques.

What are some applications of affinity chromatography?

Separating active biomolecules, isolating pure substances from low concentrations, and removing specific contaminants.

What is gel filtration chromatography?

A technique used to separate proteins based on size and shape.

What is one method used for protein concentration?

Dialysis-based methods.

What is another method for protein concentration?

Evaporation-based methods.

What is the significance of the stacking gel in a discontinuous system?

It allows for better resolution by initially concentrating the proteins before they enter the separating gel.

What is the role of ammonium sulfate precipitation in protein purification?

It is used to selectively precipitate proteins based on their solubility.

What is the elution approach in nickel affinity chromatography?

It involves using a specific ligand to elute proteins that have bound to nickel ions.

What is the purpose of desalting and buffer exchange in gel filtration?

To remove small molecules and exchange the buffer surrounding the proteins.

What is the outcome when there is no interaction between protein and stationary phase in gel filtration?

No washing step is needed.

What is the significance of determining molecular weight in protein analysis?

It helps in characterizing proteins and understanding their structure and function.

What is isocratic elution in gel filtration?

Elution occurring in Solvent A only.

What are the advantages of gel filtration?

Fastest for buffer exchange, very gentle with high yields, works in any buffer solution, removes dimers and aggregates, separates by size.

What are the limitations of gel filtration?

Limited sample volume for desalting (up to 25% column volume) and fractionation (0.5-5% column volume), narrow separation range compared to SDS-PAGE.

What is a common method for protein concentration?

Dialysis.

What are the types of dialysis-based protein concentration methods?

Reverse Dialysis (PEG precipitation), Pressure and Flow dialysis, or Ultrafiltration.

What is the purpose of gel filtration?

Buffer exchange/desalting.

What are the mechanisms of separation in chromatography?

Solubility, size and shape, hydrophobicity, charge, and biorecognition.

What are the types of chromatography based on retention mechanisms?

Gel filtration chromatography, hydrophobic interaction chromatography, ion exchange chromatography, affinity chromatography.

What is ion exchange chromatography?

A method that separates proteins based on their charge.

What are the two types of ion exchange chromatography?

Anion exchange chromatography and cation exchange chromatography.

What is the first step in ion exchange chromatography?

Binding of proteins to the solid support based on charge.

How is the charge on proteins determined?

By the concept of pI (isoelectric point).

What effect does pH have on protein elution patterns in ion exchange chromatography?

It influences the charge of proteins and their interaction with the ion exchange resin.

How do cation exchange and anion exchange chromatography differ?

Cation exchange attracts positively charged molecules to a negatively charged support, while anion exchange attracts negatively charged molecules to a positively charged support.

How are bound cations eluted in cation exchange chromatography?

By exchanging them with other cations.

How are bound anions eluted in anion exchange chromatography?

By exchanging them with other anions.

What is gradient elution in ion exchange chromatography?

A technique that uses salt concentration gradients to elute proteins by increasing the concentration of the eluent anion.

What are the advantages of ion exchange chromatography?

Controlled separation by changing pH, salt concentration, and/or ion exchange media; it can serve as a concentrating step; offers high selectivity.

What are the disadvantages of ion exchange chromatography?

Costly equipment and more expensive chemicals.

How does the pI of a protein influence chromatography?

It determines the buffer system and column selection for ion exchange chromatography.

What is the role of salt concentration gradients in ion exchange chromatography?

They help to elute proteins by competing with analytes for sites on the resin.

What is the principle behind hydrophobic interaction chromatography (HIC)?

HIC exploits the hydrophobicity of proteins, where proteins are bound to a column at high salt concentrations and eluted with a gradient of decreasing salt concentration.

What is the hydrophobic effect in relation to protein solubility?

The hydrophobic effect refers to the tendency of non-polar molecules to associate with each other in water, reducing the entropy of water molecules around them.

What does 'salting out' refer to in protein purification?

Salting out is the process where proteins become less soluble at high salt concentrations, allowing for their precipitation and fractionation.

What are the advantages of using hydrophobic interaction chromatography for protein purification?

Advantages include less structural damage to proteins, the ability to load large sample volumes, compatibility with high ionic strength samples, and elution with low salt.

What are the two types of hydrophobic chromatography mentioned in the notes?

Reverse Phase Chromatography and Hydrophobic Interaction Chromatography (HIC).

What is the role of ammonium sulfate in protein purification?

Ammonium sulfate is used to precipitate proteins by increasing salt concentration, which can help in fractionating and purifying proteins.

What happens during the binding step in hydrophobic interaction chromatography?

Proteins are loaded onto the column at high salt concentrations, where strong hydrophobic interactions occur.

What is the elution process in hydrophobic interaction chromatography?

Proteins are eluted from the column by gradually decreasing the salt concentration.

What is the significance of the hydrophobicity of proteins in chromatography?

Hydrophobicity influences how proteins interact with the chromatography medium, affecting their retention and separation.

What is the definition of biorecognition in the context of chromatography?

Biorecognition refers to the specific interactions between biomolecules that can be utilized in affinity chromatography.

What are the general steps involved in liquid chromatographic separations?

Preparation of solutions, loading samples, binding, washing, elution, and analysis of fractions.