Lecture 8: Purification Of Protein-based Pharmaceuticals Flashcards
Describe generalised downstream processing.
After upstream processing and protein production, the downstream process begins.
- If the product of interest is Intracellular, the cell must be disrupted (by chemical, mechanical, enzymatic or physical means). If the protein is extra cellular, then no disruption is necessary.
- Separation of solids from liquids: centrifugation/sedimentation, and extraction/filtration.
- Concentration of protein: evaporation, ultrafiltration, adsorption and precipitation.
- Purification: chromatography
- Formulation: freeze/spray drying, sterile filtration
- Final product
How are proteins usually serperated in the process of purification?
Proteins are typically serperated from each other based on size and charge.
What are some techniques used to achieve microbial cell disruption?
Treatment with chemicals: detergents, antibiotics, solvents Exposure to alkaline conditions Homogenisation Agitation in the presence of abrasives Treatment with lysozyme
Describe the centrifugation and sedimentation of proteins in the purification process.
Crude extract prepared from protein source (usually tissue or microbe) and centrifuged to separate cells, organelles and macromolecules.
Sedimentation is dependent on the size, shape and density of the particle, and the density of the solution being centrifuged.
Examples of centrifugation include differential centrifugation and density gradient centrifugation.
Describe differential centrifugation.
Tissue sample is homogenised, and the homogenate undergoes a series of centrifugations at increasing speed.
Low speed centrifugation pellet contains whole cells, nuclei, cytoskeleton so and plasma membranes.
Supernatant undergoes medium speed centrifugation. Pellet contains mitochondria, lysosomes and peroxisomes.
Supernatant undergoes high speed centrifugation. Pellet contains microsomes (ER fragments) and small vesicles.
Supernatant undergoes very high speed centrifugation. Final pellet contains ribosomes and large macromolecules, while supernatant contains soluble proteins.
Describe density gradient centrifugation.
Also known as isopycnic (sucrose-density) centrifugation.
Sample is added to a sucrose gradient and then centrifuged. The most dense components move lower through the gradient than the lighter components. The sample is then fractionated to seperate the components of different densities.
Describe protein precipitation (salting out).
Increasing the salt concentration increases the solubility of globular proteins by reducing the electrostatic attractions between proteins that would otherwise lead to protein precipitation.
But, if the salt concentration exceeds 1M, the salt competes for water molecules and so the protein precipitates out of solution.
Describe separation of proteins by dialysis.
A sample is placed in a semipermeable membrane in a bathing solution.
Small molecules diffuse through the membrane and into the bathing solution, while the proteins remain.
Describe ultrafiltration.
Proteins in solution are centrifuged through a porous membrane. Depending on the size of the pores, different sized molecules, ions and/or proteins will either pass through, or remain.
Summarise protein purification in general.
Protein purification utilises characteristics that distinguish the protein of interest from other protein molecules. Such characteristics include:
Size and shape
Overall charge
Presence of surface hydrophobic groups
Ability to bind various ligands
All these properties depend on the sequence of amino acids in the protein.
Describe the characteristics of amino acids in aqueous solution at various pH.
At neutral pH, amino acids are dipolar, and are known as zwitterions. This means they act as both acids (proton donors) and bases (proton acceptors) = amphoteric. They are referred to as ampholytes.
The ionisation state of amino acid varies with pH. In acidic conditions, both the carboxyl group and the amino group are protonated (carboxyl group is non-ionised, amino group is ionised). In basic conditions, both the carboxyl group and the amino group are deprotonated (carboxyl group is ionised and the amino group is unionised).
Describe the titration of amino acids.
Acid-base titration implies the gradual addition or removal of protons.
Simple mono-amino mono-carboxylic alpha-amino acids possess two groups that can yield protons (diprotic acid).
Titration of amino acids yield characteristic curves.
pK = pH at which the amino acid is half dissociated. It is a measure of the tendency of a group to give up a proton. It also indicates when an amino acid (eg glycine) has the greatest buffering power.
pK1 corresponds to carboxyl group.
pK2 corresponds to amino group.
pI: at this point on the titration curve, the first proton has been removed, and the removal of the second proton has commenced. It is the pH at which glycine is fully ionised but posesses no net charge (isoelectric point). Glycine has a net negative charge above its pI and a net positive charge below its pI.
Describe titration curve data.
Amino acids with ionisable R groups have more complex titration curves.
They have three pK values, corresponding to three ionisable groups.
The pK value depends on temperature, ionic strength and the micro environment of the ionisable group.
The pI reflects the nature of the R group.
Briefly describe how amino acids interact with water (polarity, pH etc).
Non-polar AAs: hydrophobic
Polar AAs: generally hydrophilic, but some mildly hydrophobic.
Acidic AAs: hydrophilic
Basic AAs: hydrophilic
What is a hydropathy plot?
Graphs the hydrophobicity and hydrophillicity of amino acids within a protein.
